Autotrophic CO2 Assimilation and the Evolution Ribulose … · of D-ribulose-1,5-diphosphate (RuDP)...

BACTERIOLOGICAL REVIEWS, Sept. 1973. p. 289-319Copyright 0 1973 American Society for Microbiology

Vol. 37, No. 3Printed in U.S.A.

Autotrophic CO2 Assimilation and the Evolutionof Ribulose Diphosphate Carboxylase

BRUCE A. McFADDEN'

Department of Biochemistry, University of Leicester, Leicester LEJ 7RH, England

INTRODUCTION 289

AUTOTROPHIC CO2 ASSIMILATION 290

Pathways in Plant Photolithotrophs ............................................290

Pathways in Photolithotropic and Photoorganotrophic Bacteria 290

The reductive carboxylic acid cycle ...........................................290

The Calvin cycle 292

Pathways in Chemolithotrophic Bacteria 294

Regulation of the Calvin Cycle in Bacteria 294

Control of enzyme activity: by AMP 294

By NADH ....................................................... 295

By other metabolites .......................................................295Control of enzyme synthesis ...................................................296

Control by other mechanisms 297

Comparisons with green plants 297

EVOLUTION OF AUTOTROPHIC CO2 FIXATION 298

Chemical Evolution and the First Organism, a Heterotroph ..... ................. 298Retroevolution ........................................................ 298

From Heterotrophism to Autotrophism .......... ............................... 299New Catalysts in CO.-Fixing Autotrophs ......... .............................. 300RuDP carboxylase and phosphoribulokinase ....... .......................... 300

Properties of RuDP Carboxylase............................................... 300Kinetic response to substrates .............................................. 301Metal ion requirement ....................................................... 302Specificity and mechanism of catalysis ......... .............................. 302Inhibition ............... ........................................ 303Molecular size ....................................................... 304Quaternary structure, composition, terminal and tryptic peptides .... ......... 305Immunological relationships ............................. 307

Origins of synthesis in eukaryota 307

CONCLUDINGREMARKS.............................. 308

ACKNOWLEDGMENTS.310

ADDENDUM ............................. 311

LITERATURE CITED 311

INTRODUCTIONAlthough the carbon-fixing paths reflect but

one aspect of the evolution of autotrophs, thesimilarity of these paths in all autotrophicorganisms promises insights about evolutionnot yet available from an examination of thediverse exergonic processes with which CO2fixation is coupled. Accordingly, a comparisonof these pathways and catalysis and regulationwithin them is undertaken in this article.Although the primary focus is on autotrophic

paths of CO2 fixation, the assimilation of morereduced one-carbon compounds is also brieflyconsidered in an evolutionary context. No con-

'Present address: Graduate Program in Biochemistry andDepartment of Chemistry, Washington State University,Pullman, Wash. 99163

sideration, however, is given to energy-trappingmechanisms or reverse electron flow (38) inautotrophs. For a more comprehensive treat-ment of autotrophy in prokaryotes, including itsbasis, the review of D. P. Kelly is excellent(112), as is the earlier review of the photosyn-thetic bacteria by Pfennig (167). In composingthe present review, references to meeting ab-stracts or personal communications have gener-ally been avoided. Rare exceptions involve ex-perimental work with which I am familiar.

For the sake of precision, the following no-menclature is used to describe microorganismson the basis of energy sources utilized (132).Photolithotrophy and photoorganotrophy referto the capacity for light-dependent growth re-quiring exogenous, inorganic hydrogen donors

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and organic hydrogen donors, respectively. Che-molithotrophy and chemoorganotrophy refer tothe capacity for growth in the dark that isdependent upon the oxidation of exogenous,inorganic substances and organic substances,respectively.

AUTOTROPHIC CO2 ASSIMILATION

Pathways in Plant PhotolithotrophsChemical similarities between photosynthesis

in bacteria and green plants were first recog-nized in 1930 by C. B. Van Niel and laterexpanded and summarized by him (233). Basi-cally he postulated that the two processes wereexamples of photochemical reduction of carbondioxide with different hydrogen donors, andfrom this he derived the well-known generalequation for photosynthesis: CO2 + 2H2Alight (CH20) + H20 + 2A. In the case of

green plants, H2A was H20 and 2A was there-fore 02, whereas in bacteria 2A was not 02 andH2A was variable. The demonstration withilluminated chloroplasts in 1937 by Hill (85)that ferric ion could replace CO2 as an oxidantwith resultant 02 evolution from water sepa-rated the reduction of CO2 from light-depend-ent 02 formation. Thus increased attention wasfocused upon the possibility that the reductionof CO2 was a dark reaction.There followed at Berkeley the elegant eluci-

dation of the chemistry of the dark reactions inplants that requires no detailed reiteration. Inessence, Calvin and his colleagues proved thatthe reductive pentose phosphate cycle is themain CO2 fixation path in all green plantsexamined (for a review, see 33). The first stableproduct of CO2 fixation was 3-phospho-D-glyc-erate which was derived from the carboxylationof D-ribulose-1, 5-diphosphate (RuDP) cat-alyzed by RuDP carboxylase. Other enzymesthat ostensibly also functioned uniquely inplant photolithotrophic CO2 assimilation werephosphoribulokinase, D-sedoheptulose-1,7-diphosphatase, alkaline D-fructose-1,6-diphos-phatase, and nicotinamide adenine dinucleo-tide phosphate-linked D-glyceraldehyde-3-phos-phate dehydrogenase. Among plants the wide-spread distribution of these and other catalystsin the reductive pentose phosphate (Calvin)cycle left little doubt about the ubiquity of thecycle and implied its function in these species.

It was therefore of considerable interest whenHatch and Slack (76), extending the earlierobservations of Kortshak et al. (120), providedevidence for the function of a new carboxylationpathway in sugar cane leaves. Their data sup-ported rapid incorporation of "CO2 into C-4 of

malate and aspartate and a slower incorpora-tion into C-1 of 3-phosphoglycerate. Similarobservations were made with other Gramineae(79) and in species from the families Cypera-ceae, Amaranthaceae (79, 100), Portulacaceae(77), and Chenopodiaceae (100, 161). Of partic-ular significance, however, were subsequentexperiments in which the rate of loss of radioac-tivity from the dicarboxylic acids was shown toequal the rate of labeling of 3-phosphoglyceratein maize leaves (101). Because of these andrelated observations and reports that RuDPcarboxylase was present at unusually low levelsin several of these angiosperms (100, 201),Hatch and Slack postulated a transcarboxyla-tion reaction in which C-4 of the dicarboxylicacids derived from CO2 (or HCO2-) was trans-ferred to RuDP to yield two molecules of phos-phoglycerate (201). To date, however, a majorweakness of this interpretation has been thefailure to detect such a transcarboxylation.Moreover, Bjorkman and Gauhl have reportedmuch higher specific activities of RuDP carbox-ylase in leaves of "C,-dicarboxylic acid pathwayplants" than those previously described (23). Inview of this, Hatch and Slack now consider itmuch more likely that the transfer of carbonfrom the C-4 of a dicarboxylic acid to the C-1 ofa 3-phosphoglycerate proceeds by decarboxyla-tion followed by RuDP carboxylase-catalyzedrefixation of the released CO2 (78). Recentevidence with isolated corn chloroplasts, how-ever, casts doubt on that interpretation. O'Nealet al. have established that corn plastids fix CO2at significant rates via the Calvin cycle withoutthe operation of a cycle involving the C-4compounds, malate and aspartate (158). What-ever the function of the C4-dicarboxylic acidpathway, it must be emphasized that the initialcarboxylation does not in itself account for netCO2 fixation by plants in which it occurs.Whether it indeed contributes to the concentra-tion of CO2 for utilization via catalysis by RuDPcarboxylase remains to be established, andWalker has assessed some of the evidence bear-ing on this point (238). In summary, the Calvincycle is the sole path that accounts for netreductive CO2 fixation in plants.

Pathways in Photolithotrophic andPhotoorganotrophic Bacteria

The reductive carboxylic acid cycle. Amongthe strictly photolithotrophic bacteria, Sadlerand Stanier (193) found that acetate was themost effective organic compound in elevatingcell yields of Chlorobium limicola growing inthe light on reduced sulfur compounds and CO2.The definitive studies of Hoare and Gibson on

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C. thiosulfatophilum (88) and by Hoare onRhodospirillum rubrum (87), a photoorgano-troph, first suggested the function of an unusualpathway in the assimilation of acetate. Theisotopic data are summarized with respect toglutamate biosynthesis in Table 1. In the case ofC. thiosulfatophilum, "C-bicarbonate or 14C-acetate was provided during growth for 24 h. Inexperiments with R. rubrum, washed malate-grown cells suspended in water were exposed inthe light to radioisotope for 1 min. In neitherorganism could function of the tricarboxylicacid cycle account for the biosynthesis of gluta-mate from acetate, for if it had, label from[2- 4C1 acetate would have been incorporatedfirst into C-4 but rapidly into C-3 and C-2 ofglutamate.

In 1966 Evans et al. (57) proposed a newferredoxin-dependent carbon reduction cycle inC. thiosulfatophilum. This cycle involved sev-eral enzymes of the tricarboxylic acid cycle inaddition to two new reductive carboxylationscatalyzed by pyruvate and a-ketoglutarate syn-thases, both of which required reduced ferre-doxin. One turn of the cycle resulted in the in-corporation of four molecules of CO2. All en-zymes of the cycle were apparently present inextracts although the presence of citrate lyasewas not unequivocally demonstrated. Gluta-mate contained 75% of the label after 30-s 14CO2fixation in the light by suspensions of C. thiosul-fatophilum in growth medium. Presumably theglutamate was derived from a-ketoglutarate,an intermediate in the postulated cycle. Of par-ticular interest was the fact that the labelingpattern of glutamate observed earlier by Hoareand Gibson could be accounted for by the car-bon reduction cycle although their data wereobtained with cells which had assimilated ace-tate in the presence of CO2. Unfortunately, the.kinetic experiments of Evans et al. (57) did notinclude fixation times of less than 30 s. Thusearly rates of labeling of glutamate versus

3-phosphoglycerate were not assessed, render-ing it impossible to evaluate the relative contri-butions of the reductive carboxylic acid andCalvin cycles. In earlier experiments on thesame species, Smillie et al. (203) had, in fact, re-ported the presence of all catalysts in the Caf-vin cycle.

Sirevag and Ormerod (200) more recentlyidentified some products of "4CO2 fixation by C.thiosulfatophilum. Their interpretation thatfixation occurred predominantly via the reduc-tive carboxylic acid cycle was, however, withoutconclusive support because fixation productswere analyzed after 15-min intervals of "4CO2fixation. Recently, Buchanan and colleagues

TABLE 1. Origin of glutamate carbons from acetateand bicarbonate

Carbon skeleton C. thiosulfato- R. rubrumphiluMa

C02- b* b

CHNH3 (c) cCH2 c, m mCH2 c, m mC02- (C) C

a Abbreviations: b, bicarbonate derived (b* wasimplied to be bicarbonate derived); c and m, derivedfrom the carboxyl and methyl carbons of acetate,respectively. The notation (c) reflects 15 to 30% asmuch specific radioactivity as c.

extended their studies of CO2 assimilation by C.thiosulfatophilum Tassajara (32). Using cellsgrown on 40 mM Na2CO3 as the sole carbonsource, they found that fixation products wereprofoundly influenced by the molarity of the14C02, H14C03-, plus 14C-2. At a total con-centration of 40 mM, the percentage of CO2fixed into glutamate, a-ketoglutarate, and gluta-mine was 50% at 5 s and was 28% into citrateplus isocitrate in the same interval. Both per-centages showed a negative slope when plottedagainst time, as would be expected were thesecompounds early intermediates. No incorpora-tion of "CO2 into sugar phosphate + phospho-glycerate was observed in times up to 25 s. Incontrast, at a total concentration of CO2, bicar-bonate, plus carbonate of 0.8 mM, which isninefold higher than that dissolved in H2O inequilibrium with air at a pH of 7.3 and 25 C(230), phosphoglycerate was the principal la-beled product after 5 s. Moreover, the percent-age of radioactivity incorporated into phospho-glycerate revealed the steepest negative slopewith time. Buchanan et al. nevertheless sug-gested that phosphoglycerate was formed as aresult of the function of the reductive carboxylicacid cycle, perhaps mainly because they couldnot detect RuDP carboxylase in extracts. In ourhands (F. R. Tabita and B. A. McFadden,unpublished data), RuDP carboxylase wasreadily detectable in the same strain now calledC. limicola forma thiosulfatophilum strain 6230(Tassajara) (kindly provided by N. Pfennig andgrown in the absence of organic compounds),although the specific activity was low (7 nmol ofCO2 fixed per min per mg of protein at 30 C, pH8.0, under optimal conditions) after transatlan-tic air delivery. In the face of the kinetic resultsat lower CO2 concentrations and enzyme analy-ses in two different laboratories, it seems morelikely that Chlorobium fixes CO2 via the Calvincycle, although this must be established more

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rigorously. There is even some doubt that thereductive carboxylic acid cycle contributes toCO2 fixation at the higher nonphysiological CO2concentrations used by Buchanan et al., be-cause in studies with '4C-succinate and 40 mMCO2, malate and fumarate were the earliestlabeled products and appeared much morerapidly than labeled glutamate plus glutamine(32). This is contrary to the expectation forfunction of the carboxylic acid cycle in a reduc-tive direction.Some enzymes of the reductive carboxylic

acid cycle have been detected in Chromatium D(29) and R. rubrum (30), and its function hasbeen postulated in these bacteria (57) on thebasis that "they show amino acids as the mainearly products of "4CO2 fixation". In support ofthe latter, the authors cited the work of Fuller etal. (62) and Hoare (87). In fact, experiments ofFuller et al. (62) established that both aspartateand 3-phosphoglycerate accounted for 70% oflabel fixed from "4CO2 by Chromatium D in 30 sin the light and that key enzymes of the Calvincycle were present at reasonably high levels-results in basic accord with those of TrUperwith Chromatium okenii Perty (228).

Moreover, in more complete kinetic studiesHurlbert and Lascelles (94) convincingly estab-lished that autotrophically grown ChromatiumD fixed CO2 by the Calvin pathway. Of specialsignificance, however, was the finding thatgrowth on pyruvate favored subsequent CO2fixation into aspartate and suppressed the levelof RuDP carboxylase. These results stress theinfluence of the mode of growth upon CO2-fixingpathways. In this connection, the results ofHoare showing that glutamate and malate weremajor early products of CO2 fixation were con-ducted in the light under H2 with malate-grownR. rubrum (87). The function of the reductivecarboxylic acid cycle in malate-grown R.rubrum does not even account for the labelingpattern of glutamate (Table 1) because it wouldhave resulted in the rapid incorporation of "4Cfrom carboxyl-labeled acetate into C-3 and C-4of glutamate, which was not observed. Quiterecently Dhillon and Silver characterized nico-tinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from R.rubrum grown on malate in the light and foundunder a variety of conditions that the enzymefunctioned only in the oxidative direction,which is the opposite direction from that re-quired in the reductive carboxylic acid cycle(48, 57).The function of pyruvate or phosphoenol-

pyruvate (PEP) carboxylase is required foracetate assimilation via the reductive carbox-

ylic acid cycle (57). Wild-type Rhodop-seudomonas spheroides lacks PEP synthase andcarboxylase (165). Recently Payne and Morris(164, 165) characterized a mutant of this orga-nism which was pyruvate carboxylase-less andwould therefore not grow photosynthetically onglucose or pyruvate without addition of C4-dicarboxylic acids. The mutant, however, grewphotosynthetically on acetate and CO2. Thissuggested that it could synthesize C4-dicarbox-ylic acids by a route not involving carboxylationof pyruvate or PEP and thus could not be thereductive carboxylic acid cycle.More recently, Beuscher and Gottschalk have

failed to detect citrate lyase (or adenosinetriphosphate [ATP]: citrate lyase), a key cata-lyst in the reductive carboxylic acid cycle, inextracts of C. thiosulfatophilum and R. rubrumexamined under various conditions (22). Be-cause the G6ttingen group could readily detectthe enzyme in extracts of Rhodopseudomonasgelatinosa, it seems unlikely that the former twoorganisms have a complete reductive carboxylicacid cycle.To recapitulate, some photosynthetic bacte-

ria have part of the enzymes of the reductivecarboxylic acid cycle. However, there is noevidence for function of this cycle as a majorCO2 fixing path during strictly autotrophicgrowth, nor is there compelling evidence forfunction of the complete cycle during hetero-trophic growth. The role and status of thereductive carboxylic acid cycle has also beenrecently reviewed elsewhere (176).The Calvin cycle. Kinetic studies with Rps.

capsulata, a photoorganotroph, established thatCO2 is fixed through function of the Calvincycle (210). Therefore, it was of interest thatYoch and Lindstrom reported that Rhodop-seudomonas palustris grown on CO2 or formatein the light (4,000 ft-c) photoassimilated eachsubstrate preferentially into amino acids in theshortest fixation time of 15 s. The compositionof the medium in which cells had been sus-pended was not specified. Although supportingdata were not provided, it was stated that thepercentage of total radioactivity fixed into glu-tamate as a function of time showed a negativeslope and that less than 6% of the radioactivitycould be found in any of the phosphate esters. Itwas also stated, although exact growth condi-tions were not specified, that the Calvin cyclefunctioned predominantly during fixation of"4CO2 in the light by malate-grown cells (247), aresult in accord with that obtained for Rps.isachenkoi grown on malate in the light which,however, also fixed 14CO2 rapidly into gluta-mate (50). The results of Yoch and Lindstrom

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with autotrophically grown Rps. palustris aredifficult to reconcile with those of Stokes andHoare, who provided both kinetic data forH14CO2- assimilation (via "CO2) and en-zymatic data that were compatible with thedominant function of the Calvin path in thesame species suspended in phosphate buffer(209). Unfortunately, Hoare and co-workers didnot specify the light intensity used duringgrowth or fixation experiments (175, 209), so adirect comparison of their results with those ofYoch and Lindstrom (247) is somewhat dif-ficult. Inferences that RuDP carboxylase func-tions in CO2 fixation by photoheterotrophicallygrown rhodopseudomonads (50, 247) are com-patible with the data of Lascelles, who observedincreased levels of the enzyme in both Rps.palustris and Rps. spheroides after growth inthe light in the presence of glutamate andmalate (128) in comparison with those aftergrowth with high aeration in the dark.

Rhodospirillum rubrum, a biotin-requiringorganism that may be cultured in the light onCO2 and H2 photoorganotrophically or hetero-trophically, affords an excellent organism tostudy the effect of growth conditions uponCO2-fixing paths and has been the subject ofseveral investigations. Working with cells grownon malate at unspecified light intensities,Glover et al. first established that 3-phospho-glycerate was the earliest major product of CO2fixation in the presence of H2 with either high orlow light intensities (69). Anderson and Fullerlater provided extensive information on fixationof CO2 by whole cells grown at a light intensityof 1,000 ft-c and suspended in growth media.The data suggested that: (i) the Calvin pathdominated in cells grown on CO2 and H2 (6) andwas important in cells grown on acetate in thelight (7), and (ii) glycolate was the first labeledproduct of CO2 utilization by cells grown onmalate-CO2 in the light (7). Although the mech-anism of glycolate-labeling by CO2 was notelucidated, enzymatic data were in basic accordwith the observations on CO2 fixation (8). Forexample, the specific activities ofRuDP carbox-ylase and alkaline fructose-1,6-diphosphatasewere drastically reduced by growth on malatecompared with growth on CO2 in the light,whereas values were intermediate after growthon acetate in the light.

Recently, Porter and Merrett have examinedthe influence of light intensity upon CO2-fixingpaths in acetate-grown R. rubrum (174). Withcells grown at high light intensity (835 ft-c) thespecific activity of RuDP carboxylase was 2.5-fold higher than after growth at low lightintensity (140 ft-c). Indeed, kinetic experiments

with cells assimilating acetate at high lightintensity showed that high-light-grown cellsincorporated "CO2 rapidly into phosphate es-ters in comparison with cells grown and fixing"4CO2 at low light intensities. The percentage oftotal radioactivity present in phosphate estersplotted against time gave a negative slope forhigh-light conditions versus a positive slope forlow-light conditions.

Quite recently, Slater and Morris have com-pared photoassimilation of CO2 by batch andcontinuous-flow cultures of R. rubrum grown ata light intensity of 700 to 900 ft-c. In general.cells growing on malate fixed smaller but varia-ble amounts of CO2, depending on the stage andmethod of growth. The changing rates of pho-toassimilation of carbon dioxide paralleledchanging specific activities of RuDP carboxyl-ase (202).Added bicarbonate is required for growth of

non-O2-evolving photosynthetic bacteria onfatty acids (234). It seems likely that some ofthese organisms use the fatty acids in conjunc-tion with high light intensities to provide reduc-ing power and energy for assimilation of CO2 viathe Calvin cycle. Of possible interest in thisconnection was the failure to find isocitratelyase, a key enzyme of the anaplerotic glyoxyl-ate cycle (117), in R. rubrum and Rps. spher-oides grown on acetate either in the light oraerobically in the dark, although the enzymewas present in Rps. capsulata and Rps.palustris after growth under these conditions(119).Because the blue-green algae are prokaryotic

they will be examined in the context of photoli-thotrophic and photoorganotrophic bacteria, al-though they differ from other forms tradition-ally called photosynthetic bacteria in that theyevolve oxygen during photosynthesis. In thislatter, complex property of major physiologicalsignificance, the blue-green algae are similar tolower and higher plants. Early indications thatthe Calvin cycle functioned in CO2 assimilationwere obtained with "CO2 kinetic studies (155)and the finding that the labeling pattern (155)of polysaccharide-derived glucose was similarafter fixation of "CO2 to that found for thegreen alga, Chlorella pyrenoidosa (115). Assimi-lation of CO2 received renewed attention re-cently in studies of four strains of unicellularblue-green algae. Detailed studies of "CO2fixation in the light and during a subsequentdark period after steady-state photosynthesishad been established were in complete accordwith operation of the Calvin cycle (166).

In summary, the findings discussed establishthat growth medium, light intensity, stage of

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growth, conditions of CO2 fixation, and, per-haps, the medium in which cells are suspendedall influence CO2-fixing paths in photoorgano-trophic bacteria. Nevertheless, the evidencesuggests that the Calvin cycle is the majorassimilatory pathway during growth of all pho-tosynthetic prokaryotic organisms on CO2 as themain or sole carbon source.

Pathways in Chemolithotrophic BacteriaEvidence suggesting the function of the Cal-

vin cycle during fixation of "4CO2 either byphotoreduction in the presence of H2 or bycoupling with the hydrogen-oxygen reaction inthe dark was first obtained with the algaScenedesmus (15). Making allowances for abrief dark period after fixation but before ex-traction of cells by alcohol, negative slopes wereobtained for the time course of incorporation oflabel into malate and organic phosphates con-sisting mainly of phosphoglycerate. Similar re-sults were obtained after photosynthesis by thisalga in the absence of H2. Although the datadid not rigorously rule out "C-malate as theprecursor of "4C-phosphoglycerate via turnoverthrough fumarate to label C-1 and C-4 ofmalate, results indicated that two major pathsof CO2 fixation were occurring under all condi-tions of incorporation. One involved incorpora-tion into C4-dicarboxylic acids and the otherinvolved the Calvin cycle. Thus in this alga thepaths of CO2 utilization were essentially identi-cal in spite of coupling to different reductantsand energy-trapping mechanisms. This wassubsequently confirmed in more definitive ex-periments (67). Because assimilation of CO2driven by photoreduction with H2 or the reac-tion of hydrogen with oxygen in the dark wasalso known to occur in bacteria, the need forresearch on autotrophic bacteria in that erawas evident.Among the chemolithotrophs, function of the

Calvin cycle was first established for Thiobacil-lus denitrificans (147). Intact cells fixed 14CO2most rapidly into C-1 of phosphoglycerate, andother intermediates of the cycle were labeled asexpected. In the absence of thiosulfate, cellstook up 1% as much "4CO2 (13). Subsequently,it was shown that the cycle dominated in CO2fixation by autotrophically grown Hy-drogenomonas facilis not only in the presenceof hydrogen and oxygen but also in the presenceof nitrogen and oxygen, although fixation wasreduced (137). (Davis et al. recently proposed areclassification of polarly flagellated membersof the hydrogen bacteria into the genusPseudomonas (46). It was suggested that the

peritrichously flagellated member, H. eutropha.be placed in Alcaligenes. In the final analysis,the validity of the proposal will depend upongenomic homologies. The obvious deoxyribonu-cleic acid (DNA) hybridization studies muststill be completed, including one of DNA fromH. eutropha with DNA from a type strain ofAlcaligenes (180). To maintain historical clar-ity, the generic name Hydrogenomonas hasbeen retained in the present review.)Analogous studies at that time and since have

revealed that the Calvin cycle is the major pathof autotrophic CO2 fixation in various species ofHydrogenomas (20, 86), Thiobacillusthiooxidans (221), formate-grown Pseudomonasoxalaticus OX 1 (177), Micrococcus denitrifi-cans (118), Nitrobacter winogradskvi (113) andagilis (4), and probably in Ferrobacillus ferroox-idans, now called Thiobacillus ferroxidans (49).

In all of these genera, RuDP carboxylase and,in some cases, other enzymes of the cycle havebeen demonstrated in cell-free preparationsafter autotrophic growth (4, 49, 118, 142, 143,154, 178, 196, 220, 227). Ribulose diphosphatecarboxylase has also been demonstrated inextracts of Nitrosomonas europaea (181) andthe marine chemolithotroph NitrocYstis ocea-nus (35). That it is a major protein in chemoli-thotrophic bacteria was indicated by the factthat it comprises up to 15% of the solubleprotein in autotrophically grown Hv-drogenomonas eutropha (B. A. McFadden andF. R. Tabita, in press).In H. facilis. H. eutropha, T. novellus. and T.

ferrooxidans. all facultative autotrophs, it issignificant that heterotrophic growth under cer-tain conditions drastically reduced the levels ofRuDP carboxylase (5, 143, 154, 223), althoughthe specific activity of phosphoribulokinase wasmuch less variable in H. facilis (143).Taken together, these observations made

with diverse chemolithotrophic bacteria estab-lish that the Calvin path dominates in assimila-tion of CO2 by these microbes.

Regulation of the Calvin Cycle in Bacteria

Control of enzyme activity: by AMP. In1965 Johnson and Peck discovered the inhibi-tion by adenosine-5'-phosphate (AMP) of ri-bose-5-phosphate-, ATP-dependent CO2 fixa-tion in extracts of T. thioparus (99). Theyproposed that this phenomenon afforded amechanism by which ATP could be conservedin times of "energy deprivation." Inhibition ofautotrophic CO2 fixation by AMP was subse-quently observed with cell-free preparations of

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H. facilis (142) and T. novellus (5), and evi-dence suggested that one locus of the inhibitionwas the step catalyzed by phosphoribulokinase(63, 133, 143). Although adenosine diphosphate(ADP) apparently also inhibited this step (98,143), use of adenylate kinase-free enzyme willbe required to establish this rigorously. Inhibi-tion by AMP of ribose-5-phosphate-, ATP-dependent CO2 incorporation was also observedin extracts of Chromatium D, spinach (98), andRps. spheroides (187), and probably resultedfrom the inhibition of phosphoribulokinase. Incontrast, partially purified phosphoribulokinasefrom T. neapolitanus was only slightly inhibitedby 3.5 mM AMP (136). In the face of thesevaried reports, it is possible that regulation byenergy charge (12) at this key step in thebiosynthetic Calvin cycle is of importance in the"fine control" of this cycle in most but not allautotrophic bacteria. Another step that is in-hibited by adenylate is catalyzed by 3-phospho-glycerate kinase of H. facilis (141).By NADH. A major difference in carbon

metabolism between lithotrophic bacteria and02-evolving photosynthetic species (includingblue-green algae) is the use by the former ofreduced nicotinam ide adenine dinucleotide(NADH) as the reductant of CO2 instead ofreduced nicotinamide adenine dinucleotidephosphate, as was stressed by McFadden andTu (142, 143).These workers observed ATP- NADH-

dependent CO2 fixation catalyzed by the high-speed supernatant fraction from extracts of H.facilis. This Mg2+-dependent incorporation ofCO2 was inhibited by AMP. These and otherproperties and the fact that noncyclic incorpo-ration would have required a total endogenousconcentration of acceptor(s) of 10 to 15 mol perliter (143) led to the conclusion that fixationhad occurred via the Calvin cycle. Indeed, thespecific activities for this fixation correlatedwell with that of RuDP carboxylase but notphosphoribulokinase. This interpretation waslater challenged by MacElroy et al. on the basisof the important finding that NADH was apositive allosteric effector of phosphoribuloki-nase from H. facilis (135). Considering bothlines of evidence, it is obvious that NADH mayfunction in C02-fixing extracts both stoichi-ometrically as a reductant of CO2 and as apositive effector of phosphoribulokinase. Thelatter phenomenon may certainly contribute toacceleration of carbon flow through the Calvincycle, thereby tending to lower the concentra-tion of reductant.A recent report has suggested that NADH is

also a positive effector of phosphoribulokinase

from Rps. spheroides (187), whereas NADH at3.5 mM has no effect upon the enzyme from T.neapolitanus when assayed under optimal con-ditions (136). It will be important to ascertainthe natural distribution of this stimulation byNADH among lithotrophic bacteria.By other metabolites. Because the Calvin

cycle provides intermediates for all biosynthesisin lithotrophic bacteria, the possibility of ele-ments of positive and negative control exertedthrough metabolites other than NADH andATP has been evident for some time. The firstconvincing example of this was a recent reportof the highly specific inhibition by PEP ofphosphoribulokinase from H. facilis (16). Bind-ing and resultant inhibition by PEP wasstrongly cooperative at saturating substrateconcentrations and the apparent Ki was 0.55mM. Although the phosphoribulokinase from T.neapolitanus was less sensitive to PEP, syner-gistic inhibition (83%) was observed in thepresence of 2.3 mM PEP plus 3.5 mM AMP(136). Thus PEP, which is a metabolite ofcentral importance and is almost directly de-rived from the Calvin cycle, may contribute toregulation of the cycle in lithotrophs.More recently, striking inhibition by 6-phos-

phogluconate of RuDP carboxylases (Ki's ofabout 0.1 mM) from Chromatium D, Ectothio-rhodospira halophila, an extremely halophilicphotolithotrophic bacterium, H. eutropha, Ana-cytis nidulans, and spinach has been reported(222). Similar results for the spinach enzymewere published almost simultaneously (39). Inthis connection, in Chlorella pvrenoidosa, Bas-sham and Kirk used both 32P-labeled phosphateand 'IC-labeled CO2 to show that the light-darktransition was accompanied by the appearanceof labeled 6-phosphogluconate in the dark (19).This was taken to reflect the function of theoxidative pentose phosphate cycle and indeedall enzymes of the oxidative phosphate cyclewere known to be present in isolated chloro-plasts (80). It was also demonstrated that pen-tose monophosphates could be converted toRuDP in the dark, yet isolated chloroplastscould not catalyze carboxylation of RuDP in thedark (19). On the basis of these results, it seemslikely that 6-phosphogluconate is the regulatorthat slows carboxylation in the dark in greenplants.

Phosphogluconate is also an important inter-mediate in the heterotrophic metabolism of thehydrogen bacteria. This intermediate is catab-olized through the Entner-Doudoroff path inthese bacteria (28, 71, 122). Thus the levels of"free" phosphogluconate in the cell may reflectthe function of this path and inhibit the Calvin

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cycle at RuDP carboxylase, an enzyme presentat high levels in addition to phosphoribulo-kinase in sugar-grown cells (143). It should benoted that Anacystis nidulans also has theenzymes of the pentose phosphate path (185);presumably, 6-phosphogluconate, which isknown to accumulate in the dark (166), regu-

lates RuDP carboxylase (10) in this organism as

well.Other potential loci of regulation in the Cal-

vin cycle are the steps involving the hydrolyticremoval of phosphate from the 1,7- and 1,6-diphosphates of sedoheptulose and fructose. Itis thought that each of these enzymes functionsuniquely in the Calvin cycle in plants andindeed the activities were separated some timeago (179). In bacteria the situation may bedifferent, however. Quite recently, fructose- 1,6-diphosphatase was purified to homogeneityfrom Rps. palustris that had been grown in thelight on a malate-amino acid mixture. The Vm.xfor sedoheptulose-1,7-diphosphate was 22% ofthat for fructose-1,6-diphosphate, and the Kmwas 3.6-fold greater. Unfortunately, the activitytowards these two substrates was not comparedthroughout purification (204). Nevertheless, theresults suggest that there may be only one

diphosphatase in Rps. palustris. It would beinteresting to compare the two species of thisenzyme (mol wt 65,000 and 130.000), which are

reversibly interconverted as a function of pHwith regard to catalytic specificity (206). Thesmaller, more active form is favored under more

alkaline conditions. The basis for the curiousinhibition of this enzyme by nucleoside triphos-phates (205) merits further examination as it iscontrary to the expectation for a step in a

biosynthetic sequence.Control of enzyme synthesis. Presumably,

three enzymes function uniquely in the Calvincycle during growth of lithotrophic bacteria:RuDP carboxylase, phosphoribulokinase, andalkaline fructose-1,6-diphosphatase, which mayfunction also as sedoheptulose-1, 7-diphospha-tase. Little detailed work has been done on

synthesis of the diphosphatases, although, as

mentioned previously, growth of R. rubrum on

malate-CO2 in the light drastically reduced thelevel of alkaline fructose-1,6-diphosphatasecompared with the level after growth on H2-CO2(7). Work on sedoheptulose-1,7-diphosphatasehas undoubtedly been hampered by the dif-ficulty in preparing the pure substrate (179).Nevertheless, it will be important to assess thediphosphatase activities in facultative litho-trophic bacteria to determine whether they are

due to a single enzyme and whether theirsynthesis is repressed by heterotrophic growth

as is the case for RuDP carboxylase under mostconditions.Of interest is the possibility that structural

genes specifying the structure of these proteinscomprise an operon (95) in lithotrophic bacte-ria. Coordinated control of the synthesis of thesecatalysts would be of obvious advantage. In thisconnection, ratios of specific activities of RuDPcarboxylase and phosphoribulokinase in H.facilis are quite variable after various modes ofgrowth as evident from Table 2, which wastaken from the data of McFadden and Tu (143).The data suggest, but do not prove, that thesynthesis of these enzymes is not coordinatelyregulated in H. facilis. The relevant geneticwork should be done on this and other orga-nisms. One approach would be to isolate isocit-rate lyase-negative mutants of H. facilis which,unlike their wild-type parents (139), would notgrow on acetate. The autotrophic growth ofthese mutants would presumably be stronglyinhibited by inclusion of acetate in the growthmedium, as would also be the case for thewild-type parents. From these mutants, it maybe possible to derive mutants that reveal ace-tate-resistant autotrophic growth and are there-fore constitutive for production of one or bothenzymes in question.The data in the right-hand column of Table 2

are also of interest. The evidence that thereductive, ATP-dependent fixation of CO2 byextracts occurred via the Calvin cycle has beendiscussed in an earlier section. As evident, thesedata revealed a reasonably good correlationwith the specific activity of RuDP carboxylasebut not that of phosphoribulokinase. The sup-porting arguments that the levels of RuDP

TABLE 2. Specific activities of RuDP carboxylase,phosphoribulokinase, and ATP-, NADH-dependent

CO2 fixation in H. facilis

Specific activitya

ATP-Growth substrate RuDP car- Phosphori- NADH-

boxylase bulokinase dependentCO, fixa-

tion

CO2 67 20 4.6Fructose 51 22 4.8Glucose 51 21 3.1Ribose 25 12 2.4Glutamate 2 13 0.9Lactate 10 13 1.6Succinate 2 13 0.2Acetate 1 2 0.4

a Expressed as nanomoles of product per minuteper milligram of protein.

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carboxylase in these extracts limited the rate ofCO2 fixation via the Calvin cycle have beenpresented elsewhere (143). As would be ex-pected, ribose-grown cells fixed a smaller frac-tion of "4CO2 into phosphoglycerate when sus-pended in ribose medium (140).

Recently Joint et al. (102) obtained a com-plex of alkaline fructose-1, 6-diphosphatase andphosphoribulokinase from autotrophicallygrown R. rubrum. By the criteria of isoelectricfocusing and polyacrylamide gel electrophore-sis, the complex was pure. Loss of both activi-ties with storage seemed to correlate with theappearance of two gel electrophoretic compo-nents. Although these two enzymes do notcatalyze sequential reactions in the Calvin cy-cle, isolation of the complex is almost certainlynot fortuitous. It raises the question of thepossible combination of this complex with othercomplexes, as yet unidentified, to construct anorganized array of enzymes which function inthe Calvin cycle. Moreover, it elevates interestin the regulation of alkaline fructose-1, 6-diphosphatase and phosphoribulokinase.Control by other mechanisms. The regula-

tion of RuDP carboxylase has proven to becomplex in H. facilis (123). When this organismwas cultured on fructose with high aeration,synthesis of the enzyme was totally repressed.In contrast, growth with lower aeration in iron-supplemented medium resulted in RuDP car-boxylase-rich cells until the late-log phase.Upon depletion of fructose, carboxylase activitydecayed rapidly and the decay could be trig-gered in cells harvested in the mid-log phaseand then deprived of fructose. The latter decayhad a half-time of about 20 min at 30 C. Asimilar decay was observed under certain condi-tions in extracts of fructose-grown cells but notin extracts of autotrophically grown cells. Al-though several lines of indirect evidence sug-gested that proteolysis was responsible, theapparent requirement ofATP for inactivation ofRuDP carboxylase was reminiscent of the en-zyme-catalyzed, ATP-requiring adenylation ofglutamine synthetase (for a review see 207).

Crystallization of RuDP carboxylase of lowerspecific activity and lower absorbancy ratio at280 and 260 nm from H. facilis (124) raises theintriguing possibility that this enzyme is regu-lated by AMP- (or ADP-)amplified adenyla-tion, at least during heterotrophic growth. Apossibly analogous decay of activity of thisenzyme was noted by Hurlbert and Lascelleswhen autotrophically grown cells of Chroma-tium D were transferred to pyruvate medium(94).A two- to threefold increase in total activity of

RuDP carboxylase was observed during purifi-cation of the enzyme in extracts of T. denitrifi-cans (138) and H. facilis (124). The data, whichwere obtained with dialyzed fractions, suggestthe occurrence of natural macromolecular in-hibitors in some autotrophic bacteria. Whateverthe correct interpretations of these phenomenaprove to be, inactivation of RuDP carboxylasemay contribute significantly to the regulation ofautotrophic CO2 assimilation. In this regard, itwill be important to compare the effects ofpartial inactivation upon maximal velocity andthe Km.Comparisons with green plants. The regu-

lation of CO2 assimilation by green plants hasbeen recently reviewed by Bassham (18). Exper-imental approaches discussed included the al-teration of metabolite pool sizes after achievingsteady-state photosynthesis. Perturbation hasbeen achieved by dark periods or the addition ofcompounds such as octanoate and vitamin K5.With the latter, for example, it was possible tomimic the dark-induced accumulation of 6-phosphogluconate described previously. In ad-dition, use of synchronous algal cultures wasdescribed. By use of cultures at different stagesof the division cycle and detailed studies ofmetabolite variations during the approach tosteady-state photosynthesis in each, specificsites of regulation of carbon metabolism in thelight were tentatively identified. These were (i)steps catalyzed by fructose-1,6-diphosphataseand RuDP carboxylase, (ii) the synthesis ofsucrose, probably at the stage of sucrose phos-phate synthesis, and (iii) the conversion of PEPto pyruvate. The power of these experimentalapproaches with intact cells or chloroplasts isself-evident, but as yet little has been done withthe lithotrophic bacteria along these lines.A recent observation may be relevant. Bu-

chanan and colleagues found that highly puri-fied, spinach alkaline fructose- 1, 6-diphospha-tase was strongly stimulated by a partially puri-fied protein and reduced ferredoxin (31). It wassuggested that the activity of fructose-1, 6-diphosphatase may be increased indirectly bythe level of reduced ferredoxin, thereby provid-ing a link between light and dark reactions. Itwill be important to probe for an analogousstimulation in the photolithotropic bacteria.Of particular regulatory significance may be

the effect of 10 mM Mg2+ in lowering the pHoptima of the plant but not bacterial RuDPcarboxylases from the alkaline side into therange of 6.5 to 7.0 (217, 218), if the observedshift proves to be independent of the bufferingspecies. Sugiyama et al. speculated (218) thatactivation of RuDP carboxylase by Mg2+ may

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be linked to the light-induced pH decreasewithin chloroplasts accompanied by a change inchloroplast structure (96, 162).

EVOLUTION OF AUTOTROPHIC CO2FIXATION

Chemical Evolution and the First Organism,a Heterotroph

The concept that organic compounds whichserve as the basis of life were foamed when theearth had a reducing atmosphere of ammonia,hydrogen, methane, and water vapor was firstsuggested in a booklet by Oparin in 1924,although this postulate did not become widelyknown until 1938 (159). Since then, dating fromthe experiments of Miller in 1953 which estab-lished that amino acids could be produced bypassing an electrical discharge through thisatmosphere, numerous chemical syntheses havebeen described under conditions simulatingthose of primitive earth. These have been re-viewed recently (34, 129, 172) and will not bereiterated in detail here. Suffice it to say thatamino acids, purines, pyrimidines, sugars, fattyacids, porphyrins, proteins, and obligonucleo-tides have been produced under such condi-tions. Thus it is likely that chemical evolutionproduced a complex array of compounds be-tween the formation of the earth about 4.7 x 109years ago and the appearance of the first orga-nisms approximately 1.6 x 109 years later (34,129). This interval is based in part upon recentdescriptions of unicellular algal and bacterialfossils from the Pre-Cambrian period (17, 56,197) and may, of course, prove to be shorterwith future paleo-ontographical evidence.

Oparin in 1924 (159) and, independently,Haldane in 1929 (73) were the first to postulatethe emergence of life in the presence of diverseorganic compounds that had accumulated onthe prebiotic earth, especially in the oceans.Thus it was recognized that the first livingentity probably had little or no biosyntheticcapacity.

RetroevolutionA plausible mechanism for the evolution of

catalysts in biosynthetic sequences remained anenigma until 1945. Horowitz recognized theimprobability of the development of sequencesenabling the eventual conversion of early inter-mediates to products which were required forgrowth by emphasizing that each acquired stepwould have been selectively worthless andtherefore unlikely to persist (92). As an alterna-tive, with the premise that prebiotic environ-ments contained all compounds to be utilized

eventually in biosynthesis in descendant orga-nisms, Horowitz postulated that the develop-ment of biosynthetic paths occurred by a proc-ess often referred to as retroevolution. To illus-trate, we may consider the evolutionary devel-opment of the biosynthesis of an amino acid bya pathway now known to be:

A 24 B -4 C -t D -'Ee- amino acid

It was postulated that depletion of the essentialnutrient (in this case an amino acid) by aprimitive organism accompanied by the gain ofan enzyme which catalyzed the conversion of Eto the amino acid would have conferred anenormous selective advantage upon that orga-nism. By carrying this hypothesis to its logicalconclusion, the evolution of entire biosyntheticsequences was envisaged.The hypothesis of Horowitz was later en-

larged by Lewis, who suggested that duplicationof the ancestral gene specifying the terminalcatalyst (e in the model pathway) would haveresulted in a redundant gene which might thenhave been modified to specify a protein (d in themodel pathway) that catalyzed the formation ofthe penultimate compound in the pathway(130). In its logical conclusion, retroevolution ofall structural genes in a pathway would thenhave occurred from a common ancestral origin(that specifying e) by tandem duplication andmutation of each resultant gene. This is easierto visualize when it is remembered that a givenpostulated precursor gene already specified thethree-dimensional binding site for a substratethat was to become a product of the precedingreaction in the growing pathway. Thus muta-tion had only to result in the modification of anexisting binding site and acquisition of new sitesand catalytic capacity.The views of Lewis were further refined by

Horowitz, who pointed out that the existence ofoperons-adjacent, coordinately regulated unitsof functionally related genes-accounted forthe hypothesized tandem gene duplication andmodification (93). He also emphasized thatevolution by this mechanism should have re-sulted in detectable homologies in sequentiallyfunctioning proteins specified by an operon.Hegeman and Rosenberg have recently re-viewed the limited data that bear on this point(81) and which provide no evidence for the pre-dicted homologies.There is one line of direct evidence compati-

ble with retrograde evolution. Lin and co-work-ers (131, 246) showed that xylitol transport wasgained by "fitter" mutants of A. aerogenesgrowing at the expense of xylitol after the initial

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acquisition of a dehydrogenase which catalyzedconversion of xylitol to xylulose.

In conclusion, the theory of retroevolution asoriginally promulgated by Horowitz in 1945 (92)is very attractive in accounting for the evolutionof biosynthetic paths. For a given path, theinitiating step was acquisition of the terminalcatalyst. This catalyst, and subsequently ap-pearing ones as well, might have arisen frommutation of genes specifying structures of en-zymes of similar function in other pathways.Utilization of pre-existing structural genes,however, begs the question of the origin of themost primitive biosynthetic paths. Perhapsonly in those paths will homologies betweenfunctionally related proteins be found.

From Heterotrophism to AutotrophismAccepting the reasonable premise that the

most primitive organism was a heterotroph withlimited biosynthetic capacity, we may ask howautotrophs appeared. Presumably the predeces-sors were anaerobic and the gain of assimilationof CO2 superimposed upon a heterotrophic me-tabolism was of selective value. This need nothave meant that such organisms were capableof growth under strictly autotrophic conditionsbut only that they acquired enzymes enablinguse of the Calvin cycle to complement hetero-trophic assimilatory paths. Two nonphotosyn-thetic organisms that develop anaerobically andwhich obtain energy from the oxidation ofinorganic matter are Micrococcus denitrificansand species of Desulfovibrio. Rittenberg hasreviewed experiments which suggested that D.desulfuricans utilizes CO2 and yeast extractsimultaneously when oxidizing hydrogen withsulfate (188). It will be important to examinethis species for the presence of RuDP carboxyl-ase and phosphoribulokinase. It is of interestthat the emergence of sulfate-reducing bacteriahas been dated between 2 and 3.5 billion yearsago on the basis of geochemical evidence (14).Contrary to these implications is the recentfinding that one-half of the amino acid sequenceof ferredoxin from D. gigas is similar to aportion of ferredoxin from higher plants,whereas the other half is similar to half of themolecule from several bacteria includingChromatium (225). In the latter organisms, thesecond half shows a high degree of homology tothe first, suggesting that the ancestral struc-tural gene for ferredoxin doubled during evolu-tion. These data suggest, but do not prove, thatD. gigas is more advanced than Chromatium.

With regard to M. denitrificans, strictly auto-trophic growth on H2, 02, and CO2 is accountedfor by function of the Calvin cycle (118). The

requirement of yeast extract when nitrate issubstituted for oxygen merits further examina-tion (188). If this organism can utilize both CO2and some compounds in the yeast extract dur-ing the oxidation of H2 by nitrate, it may berelatively primitive among chemolithotrophs.

Quite recently, the isolation and characteri-zation of Methanobacterium thermoautotroph-icus has been described (248). Growth of thisorganism at 65 to 70 C on H2, CO2, and mineralsrequired only the addition of vitamins. Cysteinestimulated the growth by an unknown mecha-nism. Ribose-5-phosphate-, ATP-dependentCO2 fixation was detected in extracts, althoughthe specific activity was not measured nor wasits contribution to CO2 assimilation established(J. G. Zeikus, personal communication). If thisorganism utilizes the Calvin cycle, it may berepresentative of very primitive autotrophism.In this connection, Sagan has estimated thatstabilization of the earth's mantle occurred 4.5x 109 years ago (194). Harada and Fox havesuggested that when the earth was formed, thegravitational energy was converted to thermalenergy and the temperature of the crust shouldhave increased to about 1,000 C (74). Thus, asthe surface cooled the initial temperature of thedeveloping oceans may have been elevated.Volcanic action also should have supplied ther-mal energy. Enzymes and ribosomes of thermo-philic bacteria are known to have unusualthermal stability. Of interest also are the obser-vations that as the growth temperature in-creases the nutritional requirements generallyincrease (58). To this reviewer, the evolution ofmesophiles from thermophilic ancestors is moresensible than the converse. It is easier to en-vision the gradual loss of thermal stability asthe environment cooled because, in general,macromolecules would have functioned rea-sonably effectively without mutational altera-tion during the transition. Thus random muta-tions might have gradually been fixed resultingin the selection of macro-molecules that were"fitter" in functioning at lower temperatures.In the light of these considerations, M. thermo-autotrophicus may have descended from a veryprimitive mode of autotrophism.

Other lithotrophic organisms, however, arealso facultative and anaerobic and may there-fore reflect primitive autotrophic life. These arethe photolithotrophic bacteria, and some oftheir properties have been described in anearlier section. Of particular interest in thisgroup are members of Rhodospirillum andRhodopseudomonas. They are facultative anduse the Calvin cycle for growth on C02, whichusually requires the addition of organic growth

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factors. However, these organisms are puta-tively more advanced than their anaerobicchemolithotrophic counterparts because oftheir capacity to trap radiant energy.

Finally, among the bacteria are organismswhich grow in the presence of H2 on (i) CO2, (ii)methanol plus bicarbonate, or (iii) formate plusbicarbonate. The properties of these organisms,which produce CH4 and also may grow onshort-chain alcohols and fatty acids, have beenreviewed (176). The pathway of net carbonassimilation by these bacteria, which are mem-bers of Methanosarcina, Methanococcus. andMethanobacterium. is unknown but of obviousinterest.Given that anaerobic autotrophs developed

before aerobic forms, the sequence of appear-ance of photolithotrophs which evolve 02 versuschemolithotrophs may next be considered. Mostof the latter organisms are obligately aerobicand include one or more species of Hv-drogenomonas, Pseudomonas. Nitrobacter. Ni-trosomonas. Nitrocystis. and Thiobacillus. If 02in the ancient atmosphere was of biologicalorigin, the aerobic chemolithotrophs must havedescended from 02-evolving species. Althoughthe view that 02 originated as a result ofphotosynthesis perhaps prevails (160), the alter-native of its formation from photodissociation ofwater should not be overlooked (195).

In addition to CO2, a number of one-carboncompounds varying in oxidation state frommethane to formate can be assimilated aerobi-cally by bacteria. The assimilatory paths havebeen recently reviewed (176). The three mainroutes described are (i) a serine path, (ii) a pathinvolving N-methylated amino acids, and (iii) aribose-phosphate cycle involving formaldehydefixation. The reader should consult the excel-lent review by Quayle for details (176). Quaylehas suggested a possible evolutionary develop-ment of methane utilization from ammonia-oxi-dizing lithotrophy (176). Although there is asyet no obvious relationship between these inter-esting paths of methane utilization and thatused by predominant forms of autotrophic life,further work on the enzymology of the formalde-hyde-fixing cycle, especially, is of obvious inter-est.New Catalysts in C02-Fixing AutotrophsRuDP carboxylase and phosphoribuloki-

nase. The role of enzymes which functionuniquely in the Calvin cycle has been discussedin a previous section. The two enzymes whichhave no counterparts with closely analogousfunction are RuDP carboxylase and phos-phoribulokinase. Thus a consideration of theevolution of this cycle basically reduces to a

consideration of these two catalysts. Glycolysisis widely distributed in anaerobic bacteria andglucose-6-phosphate plus 6-phosphogluconatedehydrogenases, pentose phosphate isomerases,transketolase, and transaldolase occur in some,if not most, anaerobes (245). These observationssuggest that glycolysis and the oxidative hexosemonophosphate path were established in rea-sonably primitive organisms and that catalystsfunctioning in each pathway were availableintracellularly during acquisition of the carbox-ylase and kinase.

If in a specific, ancient ecological habitat theavailability of hexoses became limiting, orga-nisms that acquired the capacity to convert CO2to hexoses via the Calvin cycle should have hada marked competitive advantage. The obviousdilemma in this conjecture is that the acquisi-tion of both RuDP carboxylase and phos-phoribulokinase would have been required toclose the cycle. If, however, one of these twocatalytic activities already resided in anotherenzyme-albeit weakly-at the time of appear-ance of the second enzyme, the organism shouldstill have been competitive. In this connection,the most obvious enzyme that might haveshown some activity towards phosphoribulose isphosphofructokinase. Although the substrateof the latter enzyme is probably the furanose,whereas phosphoribulose is acyclic, it is per-haps significant that fructose-1,6-diphosphateis a good competitive inhibitor with respect toRuDP for RuDP carboxylase (see section In-hibition and reference 26). Therefore, an inter-esting precedent exists for apparent competi-tion between these cyclic and acyclic sugarphosphates. It would thus be of interest toexamine the specificity of bacterial phospho-fructokinases and phosphoribulokinases withrespect to phosphorylation of fructose andribulose phosphates. It is easy to imagine theevolution of a fitter phosphoribulokinase fromthe genome specifying phosphofructokinaseafter the establishment of RuDP carboxylase.No similarly close enzymatic analog of RuDPcarboxylase exists and it is tempting to con-sider that appearance of the carboxylase trig-gered the evolution of efficient autotrophic C02fixation.

Properties of RuDP CarboxylaseAs evident from the previous sections, discus-

sions of the sequence of appearance of auto-trophic forms of life in evolution are quitetentative. To attack the issue experimentallyrequires the examination of a property or prop-erties shared by all autotrophic species. Themost obvious single property is that of C02fixation, the primary step of which is catalyzed

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by RuDP carboxylase which is ubiquitousamong autotrophs.Much is known of the structure and function

as well as regulatory properties of this enzyme,but almost nothing is known about these prop-erties of phosphoribulokinase. RuDP carboxyl-ase was the subject of a review by Kawashimaand Wildman in 1970 (107), but considerablymore information is now available. Accordinglythe properties of RuDP carboxylase will beexamined in the next section.

Kinetic response to substrates. Concernstill persists as to whether RuDP carboxylaseactually catalyzes primary CO2 fixation becausethe Km for CO2, which is now known to be thesubstrate (45), is considerably higher (107) thanequilibrium concentrations of CO2 in the aque-ous milieu of autotrophic species. For example,Hatch and Slack recently emphasized (78) thatfor spinach RuDP carboxylase the Km for CO2 of0.45 mM (44) is too high to account for CO2fixation, because the "concentration of CO2 in asolution in equilibrium with air and at tempera-tures between 20 and 30 C would be about 8 ,mirrespective of pH." Invocation of the functionof chloroplastic carbonic anhydrase to concen-trate CO2 (78) may be fruitless because theconcentration of CO2 plus HCO3- in the chloro-plast cannot exceed the extra-chloroplastic con-centration of these species unless there is achloroplastic mechanism to pump in HCO3- (orrelated species). To this reviewer, the mostplausible explanation for the discrepancy be-tween the Km and (CO2) is that the Km does notreflect the true Km in situ. That this may wellbe the case was strongly indicated by thefinding with intact spinach chloroplasts thatthe Km for fixation of HCO3- (or CO2) wasabout 20-fold smaller than that for isolatedRuDP carboxylase and that the fixation oc-curred by the Calvin path (97). Moreover, themaximal rate of photosynthesis by these freshlyprepared chloroplasts was 63% of that of freshspinach leaves. These results suggest that thekinetic properties of RuDP carboxylase arealtered by removal from its natural environ-ment. This may be true for the bacterial en-zymes as well because they also have high Kmvalues for CO2 (107). These considerationsimply that RuDP carboxylase functions in vivoat concentrations of CO2 which are well belowthat required for saturation.As mentioned in a previous section, RuDP

carboxylase from Hydrogenomonas comprisesup to 15% of the "soluble" protein. Although ithas not been purified to homogeneity fromC02-grown R. rubrum or T. denitrificans, it canbe estimated that the carboxylase is about 6and 2%, respectively, of the "soluble" protein

from these two sources (9, 138). The enzyme isundoubtedly a major protein in chloroplasts ofhigher plants (107). For example, it is 16% of the"soluble" protein in spinach leaves (163). Thepresence of such large amounts of a catalyticprotein in leaf tissue has led to speculationabout additional functions of RuDP carboxylase(107). It seems likely to this reviewer, however,that the large amount present in this tissue andother sources as well more probably reflects itsinefficiency as a catalyst. The turnover num-bers, which are proportional to specific maximalvelocities, of RuDP carboxylase in terms ofmoles of CO2 fixed per second per mole ofcatalytic site at 30 C are 1 and 2 for theHydrogenomonas and spinach enzymes, respec-tively. This estimate assumes 12 and 8 catalyticsites per molecule of enzyme (mol wt 500,000),respectively (107, 124). These turnover numbersare 5- to 10-fold less than the smallest values inthe range of 10 to 107 tabulated in a recent book(21). The enigma is why such a comparativelyinefficient catalyst became established duringevolution, although it must be emphasized thatthe enzyme-catalyzed reaction is probably atleast 109 faster than the uncatalyzed non-enzymatic reaction (169).

It has become apparent from the contribu-tions of Akazawa and his colleagues that thebinding of substrates by RuDP carboxylase maybe weakly homotropic or heterotropic, depend-ing upon the source of the enzyme and the con-centration of Mg2+. In general, studies of theenzyme from Chlorella (217) and spinach (218)revealed positive homotropism for HCO.- orCO2 (152) binding. Added Mg2+ lowered theapparent Km or (S)0.5, i.e., the substrate con-centration at which the velocity was half-maxi-mal (121). In contrast, studies of the enzymesfrom Rps. spheroides and R. rubrum (2) alsorevealed homotropic interaction with HCO3-but no effect upon (HCO1-)o., by added Mg2+.Moreover, homotrophic interaction with RuDPthat vanished with added Mg2+ was revealed.The (S)0.5 for RuDP in the presence of addedMg2+ was about 0.2 mM, which is within thenormal range observed for enzyme from diversesources (107). Whereas cataloging of these li-gand-enzyme interactions will certainly be ofinterest in comparing RuDP carboxylases, itshould be emphasized that the interaction coef-ficients (n values in the Hill equation, seereference 11) did not in any case exceed 2 (2,217, 218), whereas with the plant enzyme thelimiting value would be 8 for infinite cooperativ-ity if it is assumed that each large subunit hasone catalytic site (see "Quaternary struc-ture etc." and reference 107). Thus the co-operativities observed were small and may be of

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minor significance in the "fine control" ofRuDPcarboxylase. However, the effect of 10 mMMg2+ in lowering the pH optima of the plantbut not bacterial enzymes from the alkaline sideinto the range of 6.5 to 7.0 (217, 218) may be ofgreater physiological significance as mentionedpreviously.Metal ion requirement. Kawashima and

Wildman have discussed the substitution ofMg2+ by Ni2+ or Co2+ for enzyme from somesources. In general, the efficacy depends uponage and purity of the enzyme. Generally, RuDPcarboxylase activity is highly dependent uponadded Mg2+ (107). Of interest, then, was thereport of Anderson and Fuller that the highlypurified enzyme from R. rubrum showed nodependence upon added Mg2+ (9), although 0.1mM Mg2+ was apparently present during assay.In those studies highly purified enzyme wastreated briefly with 1 mM ethylenediaminetet-raacetic acid (EDTA) and then filtered on gelwhich had been treated with EDTA for 3 daysand washed with "metal-free" tris(hydroxy-methyl)aminomethane buffer. The same bufferwas used for elution but unfortunately con-tained no EDTA. These studies do not rule out adependence upon Mg2+ but do suggest that theenzyme from R. rubrum either has an unusuallyhigh affinity for Mg2+ or that dissociation ofMg2+ is kinetically very slow. The Km for Mg2+is 1.1 and 1.4 mM for the higher plant andhydrogenomonad enzymes, respectively (107,124). In contrast to the results of Anderson andFuller are those of Akazawa et al., who observed50% activation of RuDP carboxylase (of un-known purity) from R. rubrum by added satu-rating Mg2+ (5 mM). As yet, there is no expla-nation for this discrepancy, although the R.rubrum enzyme is far less responsive to addedMg2+ than is the spinach enzyme (2, 218).

H2COP03-2

C=O

HCOH

HCOH

H2COP3- 2

H+

Specificity and mechanism of catalysis.Until quite recently, available evidence sug-gested that RuDP carboxylase was highly spe-cific for the substrates CO2 and RuDP. How-ever, in 1971 Ogren and Bowes established that02 was a competitive inhibitor with respect toCO2 for the soybean enzyme (156), leading tothe recognition that the enzyme actually func-tioned at higher partial pressure of 02 in thecleavage of RuDP. The products were phospho-glycolate (27), derived from the top two carbonsof RuDP, and phosphoglycerate derived fromC-3, C-4 and C-5 (N. E. Tolbert, in AlgalPhysiology and Biochemistry, in press). Accord-ingly, the name RuDP oxygenase has beensuggested for this activity (N. E. Tolbert, inpress). Although phosphoglycolate productionwas demonstrable after incubation of soybeanor corn leaf extracts with RuDP under air or 02(27), little is known of the natural distributionof phosphoglycolate generation by RuDP car-boxylases. This is of significance because ofconjecture that phosphoglycolate productionduring *photorespiration of plants is due tocatalysis by the oxygenase component of RuDPcarboxylase (156; N. E. Tolbert, in press).Although the precise role and degree of thisalternative in catalysis for RuDP carboxylase invivo remains to be established, it may account,in part at least, for the unusually large amountsfound in plants. Furthermore, a hitherto un-recognized function of RuDP carboxylase mayexplain why the enzyme is present at high levelsunder certain conditions of heterotrophicgrowth of bacteria (123, 143, 223). It will be ofobvious importance to probe for oxygenase ac-tivity among bacterial RuDP carboxylases.With regard to the mechanism of action, the

following pathway is consistent with the experi-mental data:

H2COP032 H2COPO32 H2COPO3-2I ~~~~~~ICo2C-0 C-OH 0-2C-C-OH

11 11 -

COH C-0O C=O+ HHCOH H1COH HCOH

H2COP3- 2 H2COPC,3-2 H2COPO3-2

-H, -H+

H2COP03-2 C02-

-02C-C-OH

H

H-C-OH

H2COP3- 2

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Kawashima and Wildman have recapitulated(107) the proof by Rose and colleagues (59, 151)that the CO2 addition occurs at C2 and thattritium is lost to the medium by C,-tritiatedRuDP during catalysis by RuDP carboxylase.On the basis of the finding that RuDP may alsoundergo oxygenolysis to phosphoglycolate andphosphoglycerate, oxygen and water presum-ably add to the double bond of the 2, 3-enediolintermediate such that 180 from molecularoxygen appears in phosphoglycolate, whereasthat from water appears in phosphoglycerate(N. E. Tolbert, in press).The reaction mechanism may be ordered on

the basis that preincubation of the enzyme withMg2+ followed by bicarbonate with subsequentinitiation of the reaction by RuDP yields amuch higher rate of CO2 fixation than do othercombinations and sequences of substrate addi-tion (124, 138, 171). Thus it has been inferredthat the preferred order of addition to theenzyme from Tetragonia, Hydrogenomonas,and Thiobacillus is Mg2+, C02, and RuDP.However, these experiments should be repeatedin the presence of exogenous carbonic anhy-drase since they may only reflect the timedependence of equilibration of the substrate,C02, with provided bicarbonate (44). In thisconnection it is of some interest that carbonicanhydrase has been recently detected in one ofthe thiobacilli (190). For elucidation of thekinetic mechanism, a detailed examination ofproduct inhibition patterns may be fruitful (40).

Inhibition. The anions, orthophosphate andsulfate, are competitive inhibitors with respectto RuDP for RuDP carboxylase from spinachand H. facilis although the Ki's are 4 to 10 mM(124, 163). In contrast, sulfate has no effectupon the enzyme from H. eutropha and ortho-phosphate is stimulatory with enzyme from thatsource (124) and slightly inhibitory with en-zyme from R. rubrum (9). The effects of anumber of plausible metabolic inhibitors ofRuDP carboxylase have been examined. Citrateappears to be a competitive inhibitor withrespect to RuDP (Ki = 0.047 mM) for enzymefor R. rubrum (9), although a physiological rolefor this inhibition remains to be established.3-Phosphoglycerate is a competitive inhibitorwith respect to HCO3- and noncompetitive withrespect to RuDP for enzyme from H. facilis andH. eutropha (124) and spinach (163). The Ki'swere 8 to 15 mM. In contrast, the enzyme fromR. rubrum was much more sensitive (K, = 0.3mM) and the opposite inhibition pattern wasobserved (9). The possibility that both citrateand phosphoglycerate inhibit the enzyme fromR. rubrum by complexing Mg2+ should be

re-evaluated in light of the possibility that thisenzyme requires Mg2+. In any case, the widelydifferent sensitivities of RuDP carboxylases tothese ligands may reflect two different reactionmechanisms. In this context, the failure of 1mM 6-phospho-D-gluconate to inhibit the en-zyme from R. rubrum in contrast to the strikinginhibition of enzyme from Hydrogenomonasand spinach (39, 222) may also reflect differentreaction mechanisms and active sites. As indi-cated in a previous section, it is likely thatinhibition of RuDP carboxylase by phosphoglu-conate is of physiological importance.Kuehn and McFadden examined the effect of

a variety of metabolites upon the enzyme fromH. facilis (G. D. Kuehn, Ph.D. thesis, Wash-ington State University, Pullman, 1968). Thefollowing compounds tested at 1 mM in thepresence of 20 mM HCO-, 20 mM Mg2+, and1.6 mM RuDP, (124) gave the indicated level ofinhibition: 5'-AMP, 42%; 2',3-AMP, 44%; ADP,42%; ATP, 34%; thiamine pyrophosphate, 33%;and L-glutamate, 35%. Because of the lack ofspecificity and reasonably high concentration ofinhibitors required, the observed inhibitionsmay not have physiological significance. Underthe same conditions, inhibition of 56% was ob-served by 1 mM fructose-1,6-diphosphate.Quite recently Siegel and Lane (199) have

extended earlier observations (243) which es-tablished that 2-carboxy-D-ribitol-1, 5-diphos-phate (CRDP) was a potent inhibitor of RuDPcarboxylase. In the earlier studies, it was foundthat Mg2- or Mn2+ promoted tight binding ofCRDP to the enzyme. Fifty percent inhibitionwas obtained by 0.2 ,M CRDP in the presenceof 0.88 mM RuDP. Equimolar amounts ofCRDP and metal ion were bound. The stoichi-ometry for RuDP binding was 8 mol per mol ofenzyme at low ionic strengths in the presence ofadded metal ion, and the disassociation constantwas 1 uM. Removal of tightly bound Cu2-,known to be in the ratio of 1 g-atom/mol ofenzyme (244), abolished the requirement forMg2+ (or Mn23) in tight binding of CRDP.Because no significant catalytic or structuraldifferences were found between the Cu2-saturated and Cu2+-free enzyme (243), the roleof Cu2+ remains obscure as does the unusualbinding ratio with RuDP carboxylase. Morerecently, the formation of a carboxylase-Mg2-CRDP complex that undergoes a slow, tempera-ture-dependent, conformational change hasbeen inferred (199). The resultant state wasinhibited and released CRDP extremely slowly.Of particular interest was the finding thatultraviolet difference spectra of RuDP carboxyl-ase promoted by RuDP and CRDP were closely

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similar, although Mg2+ was not present instudies with RuDP. These studies suggest thatin the absence of metal ion RuDP can induce aconformational state not unlike that corre-sponding to the strongly inhibited state elicitedby CRDP and Mg2+ at 30 C. Of interest, then,are data which suggest that preincubation ofthe enzyme with RuDP followed by addition ofmetal ion and HCO3- resulted in the lowest CO2fixation rate of all addition sequences tested(124, 138, 171). It seems likely that RuDP in theabsence of Mg2+ and CO2 can force the enzymeinto a less active configuration which is similarto that formed with CRDP.The basis of cyanide inhibition, which was

shown to be uncompetitive with respect toRuDP for the spinach enzyme (242), was pre-sumed at one time to be related to bound Cu2+(244), although it has proven to be more compli-cated than this (243). Whatever the correctexplanation, it is of some interest that RuDPcarboxylases in extracts of T. denitrificans, H.facilis and eutropha, Chromatium D, R.rubrum, and Chlorella pyrenoidosa were allinhibited by 0.4 mM cyanide, a concentrationwhich did not significantly deplete the concen-tration of RuDP by formation of the cyanhydrin(138).There has been considerable speculation

about the involvement of a sulfhydryl (SH)group in catalysis by RuDP carboxylase (see, forexample, reference 107). Certainly reagentssuch as p-hydroxymercuribenzoate (PMB), N-ethylmaleimide, iodoacetate, and HgCl2 rap-idly inactivate the pure or highly purified en-zyme from Hydrogenomonas (125; G. D. Kuehn,Ph.D. thesis, Washington State Univ., 1968),Rps. spheroides (3), soybean (26), wheat (211),and spinach (213). Of particular interest havebeen studies in Nagoya which established thatinactivation of a molecule of spinach enzymestarted only after modification of 10 of 96 SHgroups by PMB and was complete after modifi-cation of 30 SH groups (213, 219). Use of"4C-iodoacetamide established that four to fiveSH groups could be alkylated before onset ofloss of activity and that complete loss wasassociated with the blocking of eight to ten SHgroups (214). Preincubation of the enzyme withRuDP protected against inactivation by bothreagents, but the addition of bicarbonate andMg2+ resulted in inactivation by iodoacetamidethat was somewhat faster than in the absenceof RuDP (214). These results suggest that thereare reactive SH groups in the higher plantenzyme that are not required for catalyticactivity. A second class of somewhat less reac-tive SH groups is required for catalytic activity.

Presumably, this class is exposed during cata-lytic turnover but is unavailable upon bindingof RuDP in the absence of Mg2- and HCO3-.These latter observations further suggest thepresence of a configuration that is elicited byRuDP in the absence of HCO3- and Mg2- butwhich is significantly different from the catalyt-ically active species. Unfortunately, none ofthese experiments establishes whether SHgroups function directly in catalysis or evenwhether they are at active sites.Molecular size. The molecular weights and

sedimentation coefficients of numerous RuDPcarboxylases in varying states of purity havebeen estimated by several techniques. Althoughsome must be considered as tentative because ofenzyme impurity and inadequacy of techniquesemployed, the values, procedures used, andpurity of the preparations are tabulated inTable 3.One sedimentation coefficient of Sre, = 17

Svedberg units reported for impure enzymefrom T. thioparus and T. neapolitanus (134)was omitted from Table 3 because that value isnow believed to be at least 20% too high (R. D.MacElroy, personal communication). Data forthe higher plant enzyme are restricted to thosepreparations that were pure or almost pure. Fora somewhat more comprehensive tabulation ofproperties of the higher plant enzyme, thearticle by Kawashima and Wildman may beconsulted (107). Among the higher plant en-zymes, a seriously discrepant molecular weight(475,000) was obtained by Pon for the spinachenzyme of very low specific activity (170),although a larger species (mol wt 545,000) wasalso indicated. For those studies, approach tosedimentation equilibrium was used for times ofup to 40 h. In light of the low specific activityand complex quaternary structure of the en-zyme (next section), it seems likely that thepreparations studied were heterogeneous ini-tially or partially dissociated in the course oflong-term sedimentation, or both.The molecular weight of RuDP carboxylase

from Chlorella ellipsoidea of 4.7 x 105 (Table 3)may be closer to 5.9 x 105 (215). There are a fewinexplicable discrepancies in molecular weightfor the carboxylase from a given organism(Table 3). The molecular weight is reported tobe 83,000 and 120,000 for the enzyme from R.rubrum and 240,000 and 360,000 for the enzymefrom Rps. spheroides by two different laborato-ries. Nevertheless, a clear-cut division of RuDPcarboxylases into three size classes is possible:small (mol wt 80,000-120,000), intermediate(mol wt 240,000-360,000), and large (mol wt> 500,000). In the latter category are pure

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TABLE 3. Molecular weights and sedimentation coefficients of RuDP carboxylases

State of SedimentationSource enzyme coefficient Mol wt Methods of measurement Reference

purity (Svedbergs)

Purple non-sulfur bacteriaRhodospirillum rubrum Impure S20,w = 6.2 1.2 x 10' a Sedimentation, gel filtration 9R. rubrum Impure 8.3 x 10' Gel filtration 3Rhodopseudomonas spheroides Impure 2.4 x 105 Gel filtration 3Rps. spheroides Impure Srel = 14.5 3.6 x 10'5 Density gradient centrifugation 10Rps. palustris Impure Sre, = 12 3.6 x 10'5 Density gradient centrifugation 10

Chemolithotrophic bacteriaThiobacillus denitrificans Impure 3.5 x 10' Gel filtration 138Hydrogenomonas facilis Pure S20.w = 20.0 5.5 x 10'5' Sedimentation equilibrium 125H. eutropha Pure S2,.w = 20.0 5.2 x 10' Sedimentation equilibrium 125

Purple sulfur bacteriaChromatium D Impure Sre, = 18 6.6 x 105 Density gradient centrifugation 10Chromatium D Impure Sre, = 16-18 Density gradient centrifugation 114Chromatium D Pure? 5.5 x 105 Gel filtration 1

Blue-green algaeAnacystis nidulans Impure S,, = 19.5 6.6 x 10'5 Density gradient centrifugation 10Plectonema boryanum Impure Sr,1 = 16-18 Density gradient centrifugation 114

Green algaeChlamydomonas reinhardi Impure Sre, = 16-18 Density gradient centrifugation 114C. reinhardi Pure 5-5.6 x 105 Polyacrylamide gel electro- 68

phoresisEuglena gracilis Impure Sre, = 19 6.6 x 105 Density gradient centrifugation 10Chlorella ellipsoidea Pure S20.w = 19.0 Sedimentation 241C. ellipsoideac Impure S2 ,., = 18.4 4.7 x 105 Sedimentation, gel filtration 2, 217

Higher plantsSpinach Pure? S205W = 18.3 Sedimentation 110Spinach Pure S020, w = 21.0 5.6 x 105 d Sedimentation equilibrium 163Spinach Pure S20.W = 16.8 Sedimentation 241Spinach-beet Pure S020, w = 18.3 5.6 x 105" Sedimentation, sedimentation 186

equilibriumBean Pure S20.W = 16.5 Sedimentation 241Wheat Pure S20.W = 18.5 Sedimentation 211Chinese cabbage Pure S20., = 17.0 5.1 x 105' Sedimentation equilibrium 114Avena sativa Pure? S20.w = 18.2 5.7 x 105d Sedimentation, Stokes radius 208Tobacco Impure S20w = 18.3 Sedimentation 109

a Estimated from the Stokes radius determined by gel filtration with an assumed partial specific volume.'Approximated from S,/S2 = (mol wt Jmol wt2)' '3using S,, value and the molecular weight of R. rubrum RuDP carboxylase.Partial specific volume measured by sedimentation equilibrium in D2O and H2O (only for enzyme from H. eutropha) and

estimated from amino acid composition.d Partial specific volume assumed from amino acid composition.'Partial specific volume assumed.

enzymes that have been less thoroughly charac-terized from Ectothiorhodospira halophila (B.A. McFadden and F. R. Tabita, unpublisheddata), an obligately halophilic member of thepurple sulfur bacteria (Thiorhodaceae), and Ru-mex, maize, bean, and Elodea (70).Quaternary structure, composition, ter-

minal and tryptic peptides. Electron micro-scope examination of Chinese cabbage RuDPcarboxylase first revealed complex quaternarystructure in 1965 (75). More recent electronmicroscope investigations of the enzyme fromother higher plants have been summarized(107), and electron micrographs of the Chlorellaenzyme have been described (146). The resultshave established similarities between the plantenzymes but have not elucidated the numberand precise arrangement of subunits.

In 1967 Rutner and Lane (192) and Sugiyamaand Akazawa (211) dissociated the spinach andwheat RuDP carboxylases, respectively, withsodium dodecyl sulfate (SDS). The presence oftwo well-separated subunit types was revealedby polyacrylamide gel electrophoresis. Separa-tion of the two subunit types by preparativechromatography on Sephadex in the presence ofSDS enabled determination of the amino acidcomposition of each spinach subunit type (192).The reduced, carboxymethylated spinach beetenzyme was subsequently dissociated into twosubunit types in 8 M urea, and the compositionand molecular weights of each were determined(150). The molecular weights (by gel filtration)were 54,000 and 16,000, and those of the spinachsubunits which were estimated by SDS-polya-crylamide gel electrophoresis were 55,800 and

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12,000 (191). Thus it was of considerable inter-est that Kuehn and McFadden found only onegel electrophoretic component after dissociationof the H. eutropha enzyme with either urea orSDS, although dissociation was complex. Theapproximate molecular weight in either 3 to 6 Mguanidinium chloride or 8 M urea was 40,700and was 38,000 for the H. facilis enzyme in thepresence of guanidinium chloride (125).The amino acid compositions of the

Hydrogenomonas enzymes (125) are comparedwith those for spinach subunit peaks (types) 1and 2 (192) in Table 4. As is evident, thespinach subunits differ from one another espe-cially in glycine, alanine, isoleucine, tyrosine,histidine, and arginine content. Marked differ-ences between the composition of subunits 1and 2 have also been noted for the spinach beetenzyme (150) and for the enzymes fromChlorella ellipsoidea (mol wt 58,000 and 15,000;reference 215) and five species of Nicotiana (molwt 52,000 and 13,000; references 103, 108, 105).Comparisons reveal a striking similarity be-tween compositions of subunit type 1 and dis-parity between compositions of subunit type 2(107). It was of interest that the amino acidcompositions of RuDP carboxylase fromHydrogenomonas resembled that of spinachsubunit type 1 (Table 4), suggesting that cata-lytic potential would reside in that subunit.

TABLE 4. Amino acid compositions ofHydrogenomonas RuDP carboxylase and spinachRuDP carboxylase subunits normalized (in mole

ratios) with respect to phenylalanine

Source

Amino acid Hdogenomo- H. eut- Spinach Spinach

nas ropha subunit 1 subunit 2facilis

Aspartate 2.58 2.44 2.18 2.14Threonine 1.25 1.51 1.75 1.19Serine 1.00 1.16 0.81 0.77Glutamate 2.08 2.20 2.20 2.26Proline 1.08 1.29 1.13 1.56Glycine 2.00 2.02 2.10 1.15Alanine 2.08 2.44 2.16 0.86Valine 1.58 1.64Isoleucine 1.08 1.02 0.88 0.56Leucine 1.75 1.80 2.09 1.63Tyrosine 0.69 0.82 0.92 1.57Phenylalanine (1.00) (1.00) (1.00) (1.00)Lysine 0.80 0.88 1.18 1.21Histidine 0.56 0.56 0.67 0.45Arginine 1.42 1.60 1.44 0.99Half-cystine 0.58 0.69Methionine 0.68 0.76 0.42 0.46Tryptophane 0.34 0.40

Although this has not been rigorously estab-lished, it is noteworthy that most of the 8 to 10SH groups associated with catalytic activityare found on the large subunit of the spinachenzyme (212, 216).With regard to cysteine and cystine content,

the half-cystine analyses of the enzyme fromspinach beet (186), Avena sativa (208), andspinach (192) were similar, falling into therange of 0.4 to 0.5 when normalized with respectto the mole content of phenylalanine (107). Onthe basis of studies with PMB, it is likely thatall of the half-cystine in the spinach enzyme isaccounted for by readily titratable cysteineresidues, i.e., there are no cystines (213). Thereare about 96 SH groups per molecule of spinachRuDP carboxylase and a similar number in thewheat enzyme (211, 213). In contrast, the en-zymes from Hydrogenomonas have a similarhalf-cystine content in comparison with thehigher plant enzymes (0.6-0.7, when normal-ized with respect to the mole content of phenyl-alanine), but less than one-third are cysteineresidues on the basis of titrations with 5,5'-dithiobis(2-nitrobenzoate) in the presence of 8M urea (125). Of these, more than 70% areburied and become reactive only in the presenceof urea. These data suggest marked differencesbetween the oxidation states of the higher plantand hydrogenomonad enzymes and, perhaps,differences in SH environment. Perhaps thesedisimilarities are related to the apparent dif-ferences in quaternary structure.Recent studies of carboxy-terminal peptides

have also established subunit heterogeneity forthe plant enzyme (212, 215). The C-terminalresidue was val for the large subunit (type 1)from spinach and the sequence was -ala4-(val3,ser2)-leu for the large subunit from Chlorella.For the small subunit, sequences were -phe5-leu-(tyr3,thr2)-tyr1 and -ser2-val1 for spinach andChlorella proteins, respectively. Other studieshave suggested two types of subunits for RuDPcarboxylase from Chromatium D (mol wt 57,000and 12,000; reference 1), Chlamydomonasreinhardi (mol wt about 52,000 and 17,000;reference 68), and Rumex (mol wt 56,000 and18,000; reference 70).

Recently, tryptic fingerprinting was under-taken with RuDP carboxylase from spinach andfive species of Nicotiana (105, 110). Unfortu-nately, the enzyme from N. tabacum, althoughcrystalline (109), revealed a second minor com-ponent during sedimentation (105, 106, 109)and gel electrophoretic analysis was not re-ported. Moreover, the specific activities of boththe tobacco and spinach enzymes were ex-tremely low (0.05 Amol of CO2 fixed per min permg of protein at 30 C; reference 110; cf 1.42 for

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the spinach enzyme, reference 163), althoughthat of the tobacco enzyme could be somewhatincreased by storage at 25 C (106). Neverthe-less, it is of interest that the tryptic fingerprintsof subunit type 1 were reasonably similar forenzymes from all these sources whereas thosefor subunit type 2 were markedly different. Theconclusion reached from comparison of thenumber of peptides observed with the lysineand arginine content, that the large subunit(mol wt 52,000; reference 108) derived from theNicotiana enzyme was composed of two fusedhomologous peptides (105), must be re-evaluated in light of the recent finding that thenumber of peptides was almost twice (37) thatfound earlier.

Progress towards our understanding of thequaternary structure of RuDP carboxylase hasbeen limited until recently by tedious purifica-tion procedures required to obtain pure enzyme.However, the technique of centrifugation ofextracts into density gradients developed byGoldthwaite and Bogorad (70) promises accessto one-step purification of the large enzyme.The procedure has been used successfully to ob-tain pure enzyme from Hydrogenomonas, Ecto-thiorhodospira, and Chromatium D (189, 222;B. A. McFadden and F. R. Tabita, unpublisheddata). Other procedures will have to be devel-oped for purification of the bacterial enzymesof low and intermediate molecular weight.To summarize, information suggests the oc-

currence of large (L) and highly variable smallsubunits (S) with molecular weights of 52,000 to58,000 and 12,000 to 18,000, respectively, inRuDP carboxylase from green algae, higherplants, and Chromatium D. All of these en-zymes have molecular weights that are greaterthan 500,000. The large and small subunits canbe separated after dissociation by such agentsas SDS, urea, or alkali, although use of thelatter may result in complex dissociation (108).In general, the most likely stoichiometry of com-bination for these separable species is L.S8.

Nothing is known of the precise arrangementof the two subunits. The recent crystallizationof the tobacco enzymes (36, 109, 126) may,however, open the way to a better comprehen-sion of the quaternary structure. In markedcontrast is RuDP carboxylase fromHydrogenomonas which is large but may becomposed of a single type of subunit (mol wt40,000).Immunological relationships. Considerable

research has been done on the immune responsein rabbits elicited by RuDP carboxylase al-though not always with rigorously pure antigen(Table 5). Titrations of extracts with antibodyto the enzyme from H. eutropha revealed a

much closer relationship with H. H 1 than withH. H 16 in terms of RuDP carboxylase (138).This is of some interest because H. H 1 and H.H 16 were considered to be type strains of H.eutropha (46).

Other studies showed that some antigenicsites of RuDP carboxylase from H. eutrophawere also shared with the enzyme from H. facilisand R. rubrum and, probably, fromChromatium D and Chlorella pyrenoidosa. Lit-tle if any cross-reaction was observed for theenzyme from T. denitrificans (138), nor did theanti-spinach RuDP carboxylase inhibit the en-zyme from Rps. spheroides (3). A close resem-blance has been found between the enzymesfrom Chlorella ellipsoidea and spinach (146)and between enzymes from spinach and tobacco(110). A slight precipitin was obtained in doublediffusion studies of anti-spinach RuDP carbox-ylase against enzyme from Chromatium D, anda marked spur was observed with adjacentprecipitin for the homologous reaction. Datawith antibodies to the spinach and N. tabacumenzymes suggested that the enzymes shared onegroup of antigenic sites but that there was anadditional group present on the tobacco but notthe spinach enzyme (103).These results suggest that more quantitative

studies using complement fixation will be fruit-ful. For these, a judicious selection of antigenicRuDP carboxylases that span phylogenetic rela-tionships between autotrophs should be made.Efforts should also be made to obtain immuneresponses to each of both subunits if they can berecovered in reasonably native states.

Origins of synthesis in eukaryota. Studiesdescribed in 1970 first indicated that the largeand small subunits of RuDP carboxylases weresynthesized on ribosomes in different cellularcompartments. Kawashima (104) found that"4CO2 supplied for 15 min to tobacco plantsresulted in much higher specific radioactivity oflarge subunits than of small. The radioactivitywas largely recovered in eight amino acids.Criddle et al. (45) established that chloram-phenicol specifically inhibited the synthesis ofthe larger subunit of barley RuDP carboxylase.Cycloheximide exerted a primary effect uponsynthesis of the smaller subunit and influencedproduction of the larger subunit by rapidlyinhibiting total protein synthesis. Very recently,Ellis et al. (in Symposium on Nitrogen Metabo-lism in Plants, in press) established that peachloroplast preparations containing 40 to 50%intact chloroplasts incorporated labeled aminoacids into protein upon illumination. The incor-poration was reduced by inhibitors of photo-phosphorylation and D-threo chloramphenicoland lincomycin. Actinomycin D, which inhib-

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TABLE 5. Immunological relationships of RuDP carboxylases

Antibody to enzyme froma

Enzyme of Hydrogenomonas eutrophab Spinach Nicotiana tabacum

Double Inhibition Double Inhibition Doublediffusion by (138) diffusion by diffusion

Hydrogenomonas eutropha Identity (138)(autotrophic)

H. eutropha (heterotrophic) Identity 74%H. H I Identity 73%Rhodospirillum rubrum 53% 0 (2)H. H 16 Faint precipitin 40%Chlorella pyrenoidosa 30%H. facilis No precipitin 30%Chromatium D No precipitin 21% Spur (1)Thiobacillus denitrificans No precipitin 7%Rhodopseudomonas spheroides 0 (3)Chlorella ellipsoidea Spur (1) 70% (146)Spinach Identity (217) 50% (2) Spur (110)

Identity (110) 82% (146)Nicotiana tabacum Spur (110) Identity (110)

a References are given in parentheses.bAntibody to pure enzyme from fructose-grown H. eutropha (138).

ited uridine incorporation into ribonucleic acid(RNA), had no effect upon protein synthesis. Ofparticular interest was the observation that thelabel had been incorporated into several mem-brane proteins and one soluble protein whenproducts were examined by SDS-polyacryla-mide gel electrophoresis after lysing the chloro-plasts. The soluble protein, which was a majorradioactive product, could not be sedimentedby centrifugation at 150,000 x g for 1 h and ranexactly with the large subunit of RuDP carbox-ylase on SDS-gels polymerized from 8, 10, and12% acrylamide.When 35S-methionine was used for incorpora-

tion studies, tryptic peptides derived from thesoluble protein, which had a molecular weightof about 700,000, were identical to those derivedfrom the large subunit of RuDP carboxylasewhich had been labeled in vivo (R. J. Ellis,personal communication). Separation of thislabeled product from endogenous RuDP carbox-ylase would afford an unambiguous test forcatalysis by an oligomer lacking the smallsubunit. These intriguing results suggest thatthe large subunit of RuDP carboxylase can besynthesized in the light on chloroplast ribo-somes utilizing preformed messenger RNAwhich is coded for by chloroplast DNA.Using another approach, Kawashima and

Wildman have established the loci of DNAencoding the primary structure of the large andsmall subunits in higher plants. By using aunique peptide derived from the small subunit

of RuDP carboxylase from Nicotiana tabacumas a phenotypic marker, these workers estab-lished that the marker was transmitted in aMendelian manner in hybrids (111). In con-trast, a unique peptide derived from the largesubunit of another tobacco species was trans-mitted into hybrids by maternal inheritance(37).

In Chlamydomonas reinhardi, studies withinhibitors of protein synthesis and studies ofamino acid incorporation into large and smallsubunits have been less conclusive (68, 145).Either both subunits are made on the same typeof ribosome or, more plausibly, a very efficientcoupling mechanism shuts off synthesis of thecomplementary subunit when the synthesis of agiven subunit is inhibited (68).

In conclusion, the evidence establishes thatnuclear DNA and chloroplast DNA code for theprimary structures of the small and large sub-units, respectively, of RuDP carboxylase inhigher plants. Translation of the messengerRNAs probably occurs on cytoplasmic and chlo-roplast ribosomes, respectively. Of deep interestwill be the mechanism by which the smallersubunit is transferred to the developing chloro-plast to enable completion of the assembly ofRuDP carboxylase.

CONCLUDING REMARKSEchlin has discussed geochemical and paleo-

logical indications that the process of photosyn-thesis is an ancient one and that the first

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photosynthetic organisms were on this planetsome 3 billion years ago. Although' ultrastruc-tural details are lacking, these photosyntheticorganisms were probably similar to modernphotosynthetic bacteria in several ways. Within300 million years, blue-green algae were promi-nent photosynthetic organisms, and it was notuntil between 1.5 and 1 billion years ago thatthe first eukaryotic photosynthetic organismsappeared (53). There is general agreementamong botanists that green algae are the mostlikely ancestors of vascular plants because ofthe close biochemical similarities between thetwo groups. Moreover, the green algae are one ofthe few algal groups that have made the transi-tion from marine to fresh water (54).

Echlin has discussed the ultrastructural char-acteristics of photosynthetic bacteria includingblue-green algae and of the photosyntheticapparatus in eukaryotic cells (54). There aresimilarities found between membranous config-urations in old cells of blue-green algae and theprolamellar body of greening eukaryotic chloro-plasts (127). In comparison with membranoussystems of non-02-evolving photolithotrophs, ingeneral the lamellae in blue-green algae are inlarge sheets and there are no highly stackedarrays or tubular thylakoids (54). Polyhedralinclusion bodies, which are possibly analogousto those found in some chemolithotrophs (seefollowing passage), have been observed in theblue-green algae (148). In examining the ultra-structure of other photosynthetic bacteria forclues to early evolution of autotrophism, thediversity is quite striking (54). Only the greensulfur bacteria (Chlorobacteriaceae) have struc-tures in common, membrane-bound vesicles,that serve as the photosynthetic apparatus inmembers of Chlorobium (41, 42), purified Pe-lodictyon chlathratiforme (168), and Chloro-pseudomonas ethylicum (89), although there issome question as to the identity of the lat-ter organism (72). In contrast, various Thio-rhodaceae and Athiorhodaceae have membra-nous vesicles (24, 41, 47, 52, 60, 61, 83, 90, 236,240) or lamellar systems (24, 43, 51, 52, 64-66,84, 91, 182, 184, 226, 229, 235), and one memberof Thiorhodaceae, Thiococcus, has tubularmembranes (55). An increase in complexityfrom a supposedly primitive membrane systemto the more "advanced" system of large periph-eral vesicles found in the green bacteria hasbeen postulated (91). Although this appears tobe reasonable, hypotheses regarding present-day prototypes of primitive membrane systemsappear to be premature because of the widevariation in ultrastructure of the Thiorhodaceaeand Athiorhodaceae and the well-known influ-ence of growth conditions, especially light in-

tensity, upon the fine structure (54).Because all chemolithotrophic bacteria are

aerobic, the time of emergence of the firstchemosynthetic organism depends criticallyupon the time of appearance of oxygen in theatmosphere. Were the oxygen of photosyntheticorigin, most or all of these species would havebeen established after the blue-green algaeabout 2.8 billion years ago. Alternatively, theymay be more ancient if oxygen was available asa result of ultraviolet photolysis of water. Anexamination of the ultrastructure of these bac-teria reveals diversity. Hydrogenomonas facilis(82), H. eutropha (237), and Micrococcusdenitrificans (116) have no unusual internalstructure. Several studies of the thiobacillirevealed no unusual internal structure (144,183, 231) aside from polyhedral inclusion bodiesseen in T. ferroxidans (239), T. thiooxidans(144), and a marine thiobacillus (224). How-ever, a recent detailed comparative study re-vealed that these bodies were also present in T.thioparus, T. neapolitanus, and T. intermedius.In addition, lamellar bodies similar to those insome photosynthetic bacteria were occasionallyfound in T. thioparus, and a few cells of T.intermedius had paracrystalline bodies (198). Incontrast, Nitrocystis oceanus, a marine nitrifier(153), Nitrosomonas europaea (153), Nitrobac-ter agilis (153, 173), and N. winogradskyi (232)have highly lamellar internal structures. N.agilis contains polyhedral inclusion bodies (153,173), and there may be corresponding structuresin N. winogradskyi (232). Methanobacteriumthermoautrophicum also has a lamellar internalmembrane structure (249), as do the methane-utilizing bacteria, which either have stackedmembranes concentrated around the peripheryor bundles of disk-shaped membrane vesiclesdistributed throughout the cell (176). In reca-pitulation, all obligately chemolithotrophicgenera examined have some members, at least,that possess a lamellar internal structure, whichis also found in some photosynthetic bacteria,but it is difficult to discern any clear-cutrelationships or ultrastructural trends. Of spe-cial interest may be polyhedral inclusion bodiesof unknown function that are frequently ob-served in species of Thiobacillus andNitrobacter. Clearly, more research is neededon the relationship of structure and functionand the variation of fine structure with growthconditions.

Energy-trapping mechanisms in autotrophicspecies are quite diverse. Among photolitho-trophic bacteria which do not evolve oxygen asingle photosystem is utilized, whereas 02-evolving species including blue-green algaefunction with two photosystems. Olson has

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speculated about evolution of the second photo-system (157). Chemolithotrophs derive energy

from the oxidation of numerous inorganic sub-stances such as Fe2 , H2, NO2-, NH3, S203-,and S2-. Unfortunately, information about themolecular details of these oxidations is insuffi-cient to provide a circumspect postulate aboutevolutionary sequence. One important prop-

erty, however, is shared by all autotrophicspecies, and that is the assimilation of carbondioxide via the Calvin cycle. The acquisition oftwo catalysts in this cycle, RuDP carboxylaseand phosphoribulokinase, may have triggered(or corresponded with) the emergence of auto-trophic life. In concluding, I shall summarizethe properties of RuDP carboxylase in thecontext of evolution.The molecular weights of RuDP carboxylase

fall into three size categories: small (mol wt-100,000), intermediate (mol wt -300,000),and large (mol wt -500,000).

If increasing molecular weight of this enzymeis a consequence of later evolutionary develop-ment, the following sequence of evolution ofphotosynthetic forms can be inferred: Rhodo-spirillum (Athiorhodaceae) - Rhodopseudo-

(Athiorhodaceae) Chromatium and Ectothio-rhodospira (Thiorhodaceae) and 02-evolvingspecies (blue-green algae, green algae, higherplants).

Consistent with this trend is the observationthat only the large enzymes are sensitive toinhibition by 6-phosphogluconate. Presumably,the green sulfur bacteria (Chlorobacteriaceae)are more advanced than the Thiorhodaceae as

reflected by their internal structure and deepercommitment to autotrophy. It will be of interestto compare RuDP carboxylase from the greensulfur bacteria with that of Chromatium, whichis known to have quaternary structure closelysimilar to that for the enzyme from green algaeand higher plants (two subunit types of mol wt_55,000 and 15,000). It will also be of interest toexamine the quaternary structure of the enzymefrom blue-green algae and to assess quantita-tively the immunological relationships of theenzyme and subunits from selected autotrophicforms. Comparisons of tryptic fingerprints ofsubunits and, eventually, of their primarystructures will be feasible and interesting.Scrutiny of the natural distribution of phospho-glycolate production by RuDP carboxylases willalso be important.With regard to evolution of the chloroplast, if

one favors an exogenous origin the most logicalprecursor was a blue-green alga that becameendosymbiotic with a nonphotosynthetic orga-nism. Ultrastructural and biochemical informa-

tion as well as examples of possibly analogousendosymbiosis have been reviewed (54) andlend considerable credence to this theory ofchloroplast origin. Of special interest in thisconnection will be the quaternary structure ofthe blue-green algal RuDP carboxylase. If itcontains large and small subunits, which inhigher plants, at least, are encoded within thechloroplast and nucleus, respectively, it wouldsuggest that part of the genome of the progeni-tor of the chloroplast, i.e., the blue-green alga,was integrated into the DNA of the endosymbi-otic partner.

Positioning chemolithotrophic bacteriaamong evolving autotrophs remains an enigmabut only because there is a paucity of data onRuDP carboxylase from these forms. Only theenzyme from Hydrogenomonas has been thor-oughly characterized in terms of molecularproperties. It is composed of about 12 closelysimilar if not identical subunits (mol wt-40,000). In this respect it differs markedlyfrom the Chromatium and plant enzymes yetresponds similarly to 6-phosphogluconate, asdoes the enzyme from blue-green algae. On theother hand, RuDP carboxylase from T.denitrificans is of intermediate size and isinsensitive to 6-phosphogluconate (222). In thisrespect, it is closely similar to the enzymes fromAthiorhodaceae and it may be significant thatT. denitrificans is unusual among chemolitho-trophic bacteria in that it may be culturedanaerobically or aerobically. Of obvious signifi-cance will be comparisons of RuDP carboxylasefrom the chemosynthetic bacteria includingorganisms such as Micrococcus denitrificansand Methanobacterium thermoautotrophicus,if the enzyme is found in the latter organism.Also of interest will be a search for a possiblyanalogous enzyme among the methane-utilizingbacteria.

In the final analysis, it is evident that knowl-edge of the structure, function and regulation ofRuDP carboxylase will yield much informationabout the evolution of autotrophism.

ACKNOWLEDGMENTSThis article is dedicated to D. E. Atkinson and C.

B. Van Niel for revealing the fascinations of compara-tive biochemistry. It was composed at Leicester Uni-versity during the tenure of Guggenheim and Na-tional Institutes of Health Special Fellowships. I wishto thank H. L. Kornberg for many courtesies extendedduring that period. Thanks are also extended to himand J. R. Quayle, R. Tabita, J. M. Lord, and R. J.Ellis for commenting on the manuscript and to D. G.Lundgren for comments on ultrastructure of auto-trophic microorganisms.

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ADDENDUM

After this article was completed, three relevantpublications on RuDP carboxylase appeared. Twodescribed the oxygenase activity (T. J. Andrew, G. H.Lorimer, and N. E. Tolbert. 1973. Ribulose diphos-phate oxygenase. I. Synthesis of phosphoglycolate byfraction-I protein of leaves. Biochemistry 12:11-18,and G. H. Lorimer, T. J. Andrews, and N. E. Tolbert.1973. Ribulose diphosphate oxygenase. II. Furtherproof of reaction products and mechanism of action.Biochemistry 12:18-23) and the third described thesix-carbon reaction intermediate (B. Sjidin, and A.Vestermark. 1973. The enzymatic formation of acompound with the expected properties of carbox-ylated ribulose 1, 5-diphosphate. Biochim. Biophys.Acta 297:165-173). Also, my attention was drawn to astudy of the effect of energy charge upon phos-phoribulokinase (B. A. Hart, and J. Gibson. 1971.Ribulose-5-phosphate kinase from Chromatium sp.strain D. Arch. Biochem. Biophys. 144:308-321).

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