THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 1, Issue of January 5, Printed in U. S. A. pp. 303-308,1991

In Vivo Competition between Iron and Manganese for Occupancy of the Active Site Region of the Manganese-Superoxide Dismutase of Escherichia coli*

(Received for publication, July 18, 1990)

Wayne F. Beyer, Jr., and Irwin Fridovich From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively. These are designated Mnz-MnSOD, Mn,Fe-MnSOD, and Fez-MnSOD. Sub- stitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation. Moreover, replacement of iron by manganese restored enzymatic activity. Fez-MnSOD and the iron-superoxide (FeSOD) of E. coli exhibit distinct optical absorption spectra. These data indicate that the active site environments of E. coli MnSOD and FeSOD must differ. They also indicate that competition between iron and manganese for nascent MnSOD poly- peptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.

The univalent reduction of O2 to 0; is a commonplace event and the SODs provide a defense against 0; by catalyzing its conversion into O2 + HzO2 (1). Escherichia coli contains two genetically distinct SODs, one containing manganese (MnSOD) and the other iron (FeSOD) (2, 3). The sequences of these homodimeric enzymes, and of the genes which encode them, have been reported (4-7) and a high degree of homology is evident. X-ray crystallography has indicated great confor- mational similarity (8-12) and has identified the metal li- ganding residues as 3 histidines and 1 aspartate, in both cases. These, as well as other residues in the active site regions, have been totally conserved (5,9). The high degree of sequence and

* This work was supported by grants from the Council for Tobacco Research-U. S. A., Inc., the American Cancer Society, the National Science Foundation, and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

'The abbreviations used are: O;, superoxide anion radical; MnSOD, manganese-containing superoxide dismutase; FeSOD, iron- containing superoxide dismutase; HPLC, high performance liquid chromatography; SODs, superoxide dismutases; TSY, tryptic soy yeast extract; PVDF, polyvinylidene difluoride; ODs, octadecylsilyl; SDS, sodium dodecyl sulfate; CAPS, 3-[cyclohexylamino]-l-propane- sulfonic acid; PAGE, polyacrylamide gel electrophoresis; EXAFS, extended x-ray absorption fine structure; NBT, nitroblue tetrazolium; PTH, phenylthiohydantoin.

structural homology (5, 13) explains why many MnSODs and FeSODs can accommodate either Mn or Fe at their active sites, but not why only one of these metals, not the other, imparts enzymatic activity (14). A hybrid SOD containing one subunit of the FeSOD and one of the MnSOD has been found in E. coli and characterized (15). This hybrid SOD exhibited an electrophoretic mobility midway between that of the dimeric MnSOD and the corresponding FeSOD. The sensitivity of this hybrid SOD to H202 and to azide indicated that both subunits were catalytically active (15). The SOD from a few bacterial species is exceptional in that it is active with either Mn or Fe at the active site (16-22).

Both specific activity and Mn content of MnSODs have been reported to vary from preparation to preparation (2,23- 27) and the specific activities which have been reported (2,000-3,500 units/mg) are substantially less than that of the E. coli FeSOD (-5000 units/mg) (28). Mn contents of MnSOD (0.3-0.7 atoms/subunit) (23-27) are similarly lower than the Fe content of FeSOD (0.9-0.95 atoms/subunit) (28).

It appeared possible that variable replacement of Mn with Fe in MnSOD might occur in vivo and account for these apparent discrepancies. This is particularly reasonable since Fe(I1) competes with Mn(I1) during reconstitution of apo- MnSOD (14, 23) and since Fe(1I) inhibits biosynthesis of active MnSOD in E. coli (29-31). Moreover, growth of E. coli in the presence of 59Fe, followed by electrophoretic fraction- ation, revealed the presence of Fe in the MnSOD band (30).

E. coli grown in the presence of anaerobic electron sinks, such as NO; plus paraquat, accumulated an inactive, but reactivatable, form of MnSOD which was designated pro- MnSOD (32). Mn(I1) can directly reconstitute apoMnSOD, but not proMnSOD (33), and apoMnSOD loses the capacity for direct reconstitution if dialyzed against buffers which have not been freed of adventitious metals. It thus seems likely that proMnSOD is MnSOD whose active sites are occupied by a metal other than Mn.

We now report isolation and characterization, from extracts of aerobically grown E. coli, of three MnSODs, which contain different proportions of Fe and Mn, and whose specific ac- tivities parallel their content of Mn.

MATERIALS AND METHODS

Growth of E. coli and Isolation of MnSOD-E. coli B (ATCC 29682) were grown overnight in tryptic soy yeast extract (Difco) in a water bath shaker at 37 "C and at a flask volume/fluid volume of 5 . Cells were harvested by centrifugation and were stored at -70 "C until needed. Thawed cell paste was suspended in four volumes of 50 mM KP,, 0.1 mM EDTA, at pH 7.8, and was homogenized in a Waring blender. Lysis was achieved by three passes through a Gaulin homog- enizer a t 500 Kg/cm2, with chilling to 0 "C between passes. MnSOD was isolated from the E. coli lysate as previously described (2) except that MnC12 (30 mM) was used to precipitate nucleic acids in place of streptomycin (34). For comparison, the MnSOD (lot 97F6811) from

303

304 Fe and M n Compete during Biosynthesis of MnSOD E. coli was purchased from Sigma and used without further purifica- tion. E. coli FeSOD was purified as previously described (28).

Chromatography-An LKB "GTI" system, equipped with two pumps and a model 2152 controller, was used. The ion exchange columns Mono Q and Mono S were from Pharmacia LKB Biotech- nology Inc. while the gel exclusion columns TSK-SW3000 and Su- perose 12 were from SciCon and Pharmacia, respectively. Column effluents were monitored a t 280 nm.

Amino Acid Seqwnce-NH2-terminal sequences were determined with a Porton 2090-integrated sequencing system equipped with a Hewlett Packard 1090 HPLC for on-line analysis of the phenylthio- hydantoin (PTH) derivatives. Samples of -200 pmol in 10 pl of 5.0 mM KPi at pH 7.8 were applied to glass fiber discs (Porton protein supports) and were analyzed following the manufacturer's protocol (procedure 40). When proteins were sequenced from electroblots Immobilon polyvinylidene difluoride membrane, from Millipore, was used in place of glass fiber support. P T H derivatives of the amino acids were separated on a reversed-phase ODS-Cls amino quant column (Hewlett Packard) using an acetonitrile gradient.

Spectroscopy-Optical spectroscopy was done with a Beckman DU- 70 interfaced with an IBM PCXT-286, and data analysis employed the Dataleader software package from Beckman. The slit provided 1.0 nm resolution. Circular dichroism was examined at 20 "C with a Jovin-Ybon dichrograph interfaced to an Apple IIe computer. Data were taken at 0.1-nm intervals and represent the average of three scans. This instrument was calibrated with D-10-camphorsulfonic acid whose [ 8 ] 2 ~ . s = 7,800 deg cm2 dmol-I and [6]192.s/[O]2,0.s = -1.90 (35).

Metal Analyses-Fe and Mn were measured with a Perkin Elmer Zeeman 3030 atomic absorption spectrophotometer equipped with an HGA-60 graphite furnace, as previously described (28).

Amino Acid Analysis-Lyophilized samples were hydrolyzed by exposure to the vapor released from 6.0 N HCI a t 115 "C for 24 h (36). Norleucine was added as an internal standard to allow correction for loss of sample. The protein samples were placed into borosilicate glass tubes which had been cleaned with ethanolic KOH and then thoroughly rinsed with deionized water and then dried. Micropipettes, which had been calibrated gravimetrically, were used to deliver ali- quots of the protein solutions into the tubes in which they would be hydrolyzed. Since the sequence of MnSOD is known, we could cal- culate the nanomoles of protein from the nanomoles of given amino acids in the hydrolyzate. For these calculations only the values for the acid stable amino acids were used. Thus the results for Ser, Thr, Trp, Tyr, Cys, and Met were not used.

SOD Activity-SOD activity was measured by the xanthine oxi- dase/cytochrome c method (37), while protein concentrations were based upon the quantitative amino acid analysis described above.

Electrophoresis in polyacrylamide gel slabs was done with native (38) and with SDS-denatured (39) samples. Gels were stained for SOD activity by the photochemical procedure previously described (40) and for protein with Coomassie Blue R-250. Isoelectric focusing in the Pharmacia phast system employed precast gels in the pH range 3-9 or 5-8. Electroblotting and immunostaining were done with the LKB semi-dry apparatus as previously described (32, 33) with the modification that blotting was onto polyvinylidine difluoride mem- brane (Immobilon from Millipore) with a CAPS transfer buffer a t pH 11.0 (41, 42).

Sedimentation equilibrium was performed with a Beckman model E analytical ultracentrifuge as previously described (26).

RESULTS

Detection and Isolation of Several MnSODs-The classical procedure (2) for isolation of E. coli MnSOD gave a product whose specific activity was 1900 units/mg and which exhibited several bands of protein upon electrophoresis with or without SDS. Dialysis against sodium bicinate at pH 8.3, followed by ion exchange chromatography over DEAE-Sepharose CL-GB equilibrated and eluted with this buffer, separated the MnSOD preparation into two broad bands (I and 11) of which only band I was active (3,600 units/mg). Band I appeared homogeneous in terms of size, as determined by HPLC gel exclusion or by SDS-PAGE. However, PAGE in the native state revealed a slow migrating, inactive, minor contaminant whose presence was also made evident when NH2-terminal amino acid sequence analysis indicated two different amino

acids/cycle. When electrophoretic mobility was examined as a function of pH, three components were detected in DEAE- I and two of these were active, as shown in Fig. 1. Isoelectric focusing indicated that PI for the inactive DEAE-IA was 8.3 and for the active DEAE-IB and IC were 6.7 and 5.9, respec- tively.

HPLC with a Pharmacia Mono Q column allowed prepar- ative scale separation of these components, as shown in Fig. 2. The central portions of the protein peaks eluted from the Mono Q column were concentrated by centrifugal ultrafiltra- tion using an Amicon Centricon C-10. In accord with the results of activity staining (Fig. l) , DEAE-IA was inactive, while IB and IC were active. Moreover, IB and IC exhibited NH2-terminal sequences (15 residues) identical with that of MnSOD (6, 7), whereas DEAE-IA gave a distinct sequence. DEAE-IB and IC were also identical to MnSOD as indicated by Western blots of SDS-PAGE gels probed with rabbit anti- MnSOD.

A third, enzymatically inactive, MnSOD was detected in

SOD ACTIVITY

3 9 pH gradient n pl l gr;~dicnt 0

FIG. 1. Electrophoretic titration curve of DEAE-I using the Pharmacia Phast Gel system. A pH gradient of 3-9 was established by isoelectric focusing the ampoline-containing precast gel for 170 accumulated V/h a t 2 "C. The gel was rotated 90" with respect to the established pH gradient and either -1.5 pg of protein or 1.5 or 6.0 SOD units were loaded, and electrophoresis was performed for 70 accumulated V/h at 2 "C. The gels were stained for protein using Coomassie Blue R-250 or SOD activity using the nitroblue tetrazo- lium photochemical method (40).

DEAE-IB

DEAE-IA

z

I I I

0 4 8 12 16 20 Minutes

"_ -

FIG. 2. Mono Q HPLC anion exchange chromatography of MnSOD sample DEAE-I. The column and sample were equili- brated with 20 mM Tris-HCI, pH 8.0, and 50 pl of sample (0.26 mg/ ml) was applied and eluted with a gradient of 0-500 mM NaCl (in 20 mM Tris-HCI, pH 8.0) over 15 min. The flow rate was 1.0 ml/min, and the effluent was monitored a t 280 nm. %R refers to the NaCl gradient.

Fe and M n Compete during Biosynthesis of MnSOD 305

DEAE-I1 as a band on Western blots which was immunoreac- tive when probed with rabbit anti-MnSOD (data not shown). DEAE-I1 resolved into three components during HPLC on a Mono Q column, and as shown in Fig. 3 these were designated DEAE-IIA, IIB, and IIC, respectively. Of these only IIB exhibited a retention time identical to that of MnSOD during gel exclusion chromatography over a column of Superose-12. Moreover, DEAE-IIB behaved identically with MnSOD dur- ing SDS-PAGE, during immunostaining with rabbit anti- MnSOD, and during NH2-terminal sequence analysis (15 residues).

Identification of DEAE-IB, IC, and IIB-Analyses for Mn and for Fe revealed that DEAE-IB was fully loaded with Mn, that IC contained half Mn and half iron, while IIB was fully loaded with iron. Thus, IB corresponds with Mn2-MnSOD, IC with Mn,Fe-MnSOD, and IIB with Fe2-MnSOD. The enzymatic activities of these components paralleled their con- tent of Mn, which was also the case for Sigma MnSOD, as shown in Table I. The low residual activity of the Fe2-MnSOD is due to contamination with Mn2-MnSOD (see “Discussion”). When the procedure previously devised for removal and re- placement of the active site metal (14, 23) was applied to these fractions they all gave the same specific activity, as shown in Table 11. This establishes that their differences in activity, as isolated, reflected only differences in metal con-

r---lloo

0 10 20 30’ Minutes

FIG. 3. Mono Q HPLC anion exchange chromatography of MnSOD sample DEAE-11. The sample and column were equili- brated in 20 mM Tris-HC1, pH 8.5, and 50 p1 of sample (-1.0 mg/ml) was applied and eluted with a gradient of 0-500 mM NaCl (in 20 mM Tris-HC1, pH 8.5) as shown here as %B. The flow rate was 1.0 ml/ min, and the effluent was monitored at 280 nm.

tent, not in gross protein structure. Subtle differences in structure, to which our measurements might be insensitive, cannot be ruled out. The reconstituted samples also exhibited identical isoelectric points and anodic mobilities during PAGE in the native state, thus showing that the differences in these properties seen prior to resolution and reconstitution were also due only to their differences in ‘metal content. Reversible resolution of the Mn2-MnSOD did not restore full activity. Indeed, as shown in Table I1 revepible resolution in all cases gave a product whose specific activity was -60% that of the native Mn2-MnSOD. This reflects incomplete restora- tion of manganese. Thus, the manganese content after re- versible resolution was only 1.4 f 0.2 g atoms of Mn/dimeric enzyme molecule. Native PAGE of these Mn-reconstituted SCfDs revealed the presence of residual apoSOD, which mi- grhtes more rapidly than the Mn2-MnSOD or the Fe,Mn- MhSOD. The Mn-reconstituted SOD did not contain Fe so failure to achieve full loading with Mn was not due to com- petition by Fe during reversible resolution.

Circular Dichroism-The CD of E. coli MnSOD has been reported in the visible (24,27) but not in the ultraviolet. Mnz- MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD were diluted to 55 pg/ml in 3.5 mM KPi, 6.7 WM EDTA at pH 7.8. Protein concentrations in the stock solutions were based upon quan- titative amino acid analysis and upon a mean residue weight of 111.7, which was calculated from the amino acid sequence (6, 7). Gravimetrically calibrated micropipettes were used in making dilutions from these stock solutions. The UV CD spectra obtained for all of the MnSODs were identical, as shown in Fig. 4. The mean residue ellipticity at 222 nm and the single parameter equation of Chen and Yang (43) led to an estimate of -30% a-helix, which agrees only moderately wellwith the 40-50% found in the x-ray structures of the MnSODs from Bacillus stearothermophilus (9) and Thermus thermophilus (8).

Molecular Weights-The three MnSODs isolated from E. coli were examined by gel exclusion HPLC over Superose-12 equilibrated and eluted with 20 mM Tris chloride, 50 mM NaCl, pH 8.5. The void volume (V,) was measured with salmon sperm DNA and the total volume (V, ) with 0.01% acetone. Included volume (Vi) was calculated as V, - V,, while the distribution coefficient (Kd) was calculated from the expression K d = v, - v,,/v, - vi (44), where v, is the elution volume. Each of the three MnSODs eluted in the form of a single, symmetrical peak whose Kd was 0.46 f 0.05 and which contained 90-95% of the optical density applied. The identity of Kd for all three MnSODs indicated identity of radius of gyration and the symmetry of the elution peak indicated the absence of a detectable dissociation + reassociation equilib-

TABLE I Comparison of the iron and manganese contents of purified MnSOD samples determined using Zeeman graphite

atomic absorDtion

Sample Specific Activitf Mn/Subunitb nmol Mn‘ Fe/Subunitb Mn + Fe/Subunit ‘OD units/nmo1 Mn unitlmg

Mn2-MnSOD 4.63 f 0.19 X lo3 1.14 f 0.06 49.47 f 2.51 (0.01 Mn,Fe-MnSOD 2.09 f 0.08 X lo3 0.58 f 0.02 25.32 f 0.87 0.43 f 0.03

1.14 ? 0.06 94 f 7

Fe2-MnSOD 1.42 f 0.07 X 10’ 1.01 f 0.04 82 f 4

50.01 C0.44 1.19 ? 0.05 1.19 ? 0.05 NCd Sigma MnSOD 3.93 f 0.26 X 103 46.72 f 0.17 NDd 84 f 5

SOD activity was measured using the xanthine/xanthine oxidase cytochrome c method (33). Protein was determined from quantitative amino acid analysis and a subunit molecular weight of 22,900 g/mol

The metal content and a subunit molecular weight of 22,900 was used for this calculation; 1 mg MnSOD =

NC, not calculated; ND, not determined.

(6, 7) was used. The data represent the mean f standard deviation for four measurements.

43.66 nmol subunits.

306 Fe and M n Compete during Biosynthesis of MnSOD TABLE I1

Reversible resolution of MnSOD samples Mn2-MnSOD, Fe,Mn- MnSOD, Fe2-MnSOD

Numbers represent the mean f standard deviation for four measurements.

before reconstitution after reconstitution' of activitv Specific activity Specific activity % Recovery

unit/% unitlmg Mn2-MnSOD 4.63 f 0.19 X 10' 3.00 f 0.11 X 10' 60.8 Fe,Mn-MnSOD 2.09 -C 0.08 X 10' 3.07 2 0.06 X 10' 146.9 Fe2-MnSOD 1.42 f 0.07 X lo2 3.11 f 0.14 X 1,800.0 Sigma MnSOD 3.93 f 0.26 X 10' 3.17 f 0.29 X lo3 80.7

Resolution and reconstitution was achieved by overnight dialysis a t 4 "C (at 200 pg/ml) against 200 ml of 5.0 mM Tris, 0.1 mM EDTA, 2.5 M guanidinium chloride, 20 mM 8-hydroxyquinoline, a t pH 3.8, followed by 24 h of dialysis against several 2.0 liter changes of 5 mM Tris, 0.1 mM MnC12 at pH 7.8. Unbound Mn(I1) was then removed by dialysis against changes of 5.0 mM Tris, 0.1 mM EDTA, pH 7.8.

- 1.5l I I I 200 210 220 230 240

Wavelength ( n m )

FIG. 4. Comparison of the ultraviolet circular dichroism spectra of Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD. The proteins were present a t -55 pg/ml in 3.5 mM KP, 6.7 p M EDTA, pH 7.80. A mean residue weight of 111.7 was used to calculate the mean residue ellipticity ( [ B ] ) . Protein concentrations were determined from quantitative amino acid analysis.

rium, whch agrees with previous studies which examined the sedimentation equilibrium of apo and holoMnSOD (26). The Fe2-MnSOD was examined by sedimentation equilibrium. The graph of In AZmnm as a function of the square of the distance from the center of rotation yielded a straight line from whose slope we calculated a molecular weight of 47,038 & 264, which is in good agreement with the value of 45,800 calculated for the apoprotein from the amino acid sequence (6, 7).

Electrophoretic Mobilities-Replacement of manganese by iron, at the active site, affected the electrophoretic mobility of the protein, even though the foregoing studies indicated no differences in radius of gyration. Nevertheless, the three MnSODs, containing Mn2, MnFe, or Fen, do differ in anodic mobility, as shown in Fig. 5. Thus, Mn2-MnSOD exhibited the slowest anodic mobility while Fe,-MnSOD was the fastest and Mn,Fe-MnSOD was intermediate. These differences in anodic mobilities of the variously substituted MnSODs were also evident when immunoblots of the native gels, prepared by the method of Matsudaira (41, 42), were examined. Al- though redundant in the context of isolated proteins, this immunoblotting procedure will be useful in detecting the presence of the several forms of MnSOD in crude extracts of E. coli grown under a variety of conditions. ApoMnSOD, when prepared with Chelex-100-treated buffers to prevent inad- vertent loading with iron or other extraneous metals, was reconstitutable at neutral pH by direct addition of Mn(I1) salts. This apoMnSOD exhibited anodic mobility virtually identical to that of the Fe2-MnSOD, whose PI was 5.6. It thus

PROTEIN SOD ACTIVITY

1 2 3 4 5 6 7 8

FIG. 5. Native gel electrophoresis of MnSOD samples. Mn2- MnSOD (lanes I , 5), Mn,Fe-MnSOD (lanes 2, 6). Fe2-MnSOD (lanes 3, 7), and Sigma MnSOD (lanes 4, 8) were separated using 7.5% acrylamide gels. Lanes 1-4 contained -10 pg of protein/lane and were stained with Coomassie Blue R-250, while lanes 5-8 contained -1.5 SOD units and were stained for SOD activity using the nitroblue tetrazolium photochemical method (40).

0 240 272 304 336 368 400

Wovelength Inml

I I b.,,

FIG. 6. Comparison of the UV absorption profiles of Mnz- MnSOD (line 4), Mn,Fe-MnSOD (line 2), Fe2-MnSOD (line I ) and Sigma MnSOD (line 3). The samples were present in 50 mM KP,, 0.1 mM EDTA, pH 7.8, and the protein concentrations were normalized to 1.0 mg/ml using Dataleader software and the appro- priate factor determined from quantitative amino acid analysis.

appears that binding of iron to apoMnSOD does not raise its PI, whereas binding of manganese does so.

Absorption Spectra-Although qualitatively similar, the UV spectra of the three MnSODs differed quantitatively, as shown in Fig. 6. Thus, E, (2mnm) for Fe2-MnSOD, Fe,Mn- MnSOD, and for Mn2-MnSOD was 9.19,8.95, and 8.56 X lo4 M" cm", respectively. Increase in iron content was thus associated with an increase in E,,, (2mnm). Given a subunit weight of 22,900 and 11 Phe, 7 Tyr, and 6 Trplsubunit one may calculate (45) an E, (2wnm) of 8.61 X lo4 M" cm", which agrees very well with the measured value of 8.56 X lo4 M" cm-1 for the Mn2-MnSOD. The increase in E, (2mnm) with increase in iron content may be due to a Fe(II1)-ligand charge transfer, whose consequences can be seen more clearly on an expanded scale in the near ultraviolet (Fig. 7). When apo- MnSOD was similarly examined it was found to be transpar- ent in the region 350-400 nm, as is the Mn2-MnSOD.

Absorbance in the visible region reflects the manganese content, as shown in Fig. 8. It appears that the ratio A4,3,,,,,/

(Table 111) could be used as an index of the purity of MnSOD and more specifically, of its manganese content. E, (473nm) is 610 M" cm" when calculated on the basis of the molarity of bound Mn. The literature values for this extinc- tion coefficient range from 200 to 1700 M" cm" (27) and probably reflect errors in the measurement of [protein], of [Mn], and variations in the purity of the sample being ana- lyzed. Our value agrees best with the 530 M" cm" reported for the MnSOD from human liver (46).

Fe and Mn Compete during Biosynthesis of MnSOD 307

FIG. 7. Far UV absorption comparison of Mnz-MnSOD (line 3), Mn,Fe-MnSOD (line 2), and Fez-MnSOD (line I). The data from Fig. 6 were expanded in the 300-400 nm region to illustrate the iron-dependent transition present in Fez-MnSOD.

FIG. 8. Comparison of the visible absorption profiles of Mns-MnSOD (line 2), Mn,Fe-MnSOD (line 3), Fez-MnSOD (line 4), and Sigma MnSOD (line I). The spectra were measured in 50 mM KPi buffer, pH 7.8, containing 100 NM EDTA. A molecular weight of 45,800 was used to calculate the molar extinction coefficient.

TABLE III

Summary of visible absorption data for MnSOD samples Mn2- MnSOD, Fe,Mn-MnSOD, Fe,-MnSOD and Sigma MnSOD

The molar extinction coefficient was calculated based on the man- ganese content that was determined from atomic absorption analysis. Samples were dissolved in 50 mM KP,, pH 7.8, containing 100 PM EDTA.

Sample Mn/subunit

Mm-MnSOD Fe,Mn-MnSOD Fe*-MnSOD Sigma

M-1 em-

1.14 f 0.06 605.9 z!z 26.2 2.13 0.58 + 0.02 610.9 + 30.9 0.86

10.02 NA NA 1.07 + 0.01 618.1 f 18.7 1.45

a NA, not applicable.

Circular dichroism, HPLC gel exclusion chromatography, immunoblotting of native or SDS-PAGE gels, and sedimen- tation equilibrium revealed no detectable differences in con- formation among these three MnSODs. Moreover reversible resolution, with manganese loading, converted all three into a species whose specific activity was 3,000 units/mg. It follows that Mn2-MnSOD, Mn,Fe-MnSOD, and Fez-MnSOD differed in metal content, but not detectably in the structure of the protein. These three forms of MnSOD are probably generated by competition between manganese and iron for binding to nascent MnSOD polypeptide, within E. coli. Similar compe- tition between these metal cations for reconstitution of apoMnSOD has been seen in vitro (23). It is likely that the wide range of specific activities and of manganese contents which have been reported for E. coli MnSOD reflect variable admixtures of the Mns-, Mn,Fe-, and Fez-MnSODs in the preparations studied (2, 23-27).

The absorption spectrum of Fe,-MnSOD differs substan- The three MnSODs found in E. coli exhibited differences tially from that of E. coli FeSOD, which contains the same in isoelectric points and therefore in anodic mobility, such amount of iron (Fig. 9). This result appears surprising in view that insertion of iron, but not of manganese, lowered the p1 of crystallographic data which indicated identical liganding and correspondingly increased anodic mobility. This will be groups and ligand geometries for MnSOD and FeSOD (8-12). very useful, in conjunction with immunoblotting, in detecting The difference shown in Fig. 9 does not reflect differences in and distinguishing among these MnSODs in crude extracts of the valence of the bound iron since both Fe*-MnSOD and E. coli grown under a variety of conditions. The manganese, FeSOD, when denatured in the presence of ferrozine, only in resting MnSOD, is known to be trivalent, and we have gave the purple Fe(II)-ferrozine complex after a reductant used denaturation in the presence of ferrozine + reductant to had been added. It follows that both Fe*-MnSOD and FeSOD show that the iron in Fez-MnSOD is also trivalent. Why then contained trivalent iron. The shoulder at 350 nm, evident in should replacement of Mn(II1) by Fe(II1) lower the PI? One the spectrum of FeSOD, has been attributed to a metal-ligand possible explanation is that bound Fe(II1) lowers the pK, of a charge transfer transition (47). It should be noted that the ligated water molecule, while bound Mn(II1) does not. Alter- absorption spectrum of Fez-MnSOD shown in Fig. 9 resembles that previously seen with HzO,-inactivated FeSOD (28).

natively, the ligand environment of the two metals may be dissimilar.

DISCUSSION

Aerobically grown E. coli contain three MnSODs, which have now been isolated and characterized. The first of these,

The structures inferred from x-ray crystallographic analysis indicate identical ligand environments at the active sites of MnSOD and FeSOD (a-12). This would lead to the expecta- tion that these enzymes should be active with either Mn(III)

FIG. 9. Comparison of the far UV absorption of E. coli FeSOD (line I) and Fez-MnSOD (line 2). The spectra were measured in 50 mM KP, buffer, pH 7.8, containing 100 pM EDTA. The molar extinction was calculated using a protein molecular weight of 42,200 for FeSOD (5) and 45,800 for Fez-MnSOD (5).

designated Mn2-MnSOD has a specific activity of 4,700 units/ mg and contains two atoms of Mn/dimeric molecule. The second, designated Mn,Fe-MnSOD exhibited a specific activ- ity of -2,100 units/mg and contained one atom of Mn and one of Fe/dimer. The third, (Fe*-MnSOD) was essentially inactive (-140 units/mg) and contained two atoms of Fe/ dimer. The Mn,Fe-MnSOD is not the same as the hybrid SOD, which contains one subunit of MnSOD and one of FeSOD. This can be stated with confidence because the NH*- terminal sequences of all three MnSODs in this study were identical and in full agreement with the known MnSOD sequence (6,7) and because the anodic mobihty of the hybrid SOD differs markedly from that of any of the MnSODs (15).

308 Fe and Mn Compete during Biosynthesis of MnSOD

or Fe(II1) a t their active sites and this is not the case. The E. coli MnSOD is active with Mn(III), but not with Fe(III), at its active sites. Indeed, the small activity here reported for the Fez-MnSOD (148 units/mg) is due to a contaminant of Mn2-MnSOD rather than to a real, if tiny, activity of the Fez- MnSOD. This is known because duplicate PAGE of the Fez- MnSOD, followed by staining for activity in one case and for protein in the other, shows that the activity in Fez-MnSOD migrated to the position expected for Mnz-MnSOD whereas the bulk of the protein was at the position expected for Fez- MnSOD.

The identity of the ligands and the congruence of their geometries, revealed by x-ray crystallography, would also lead to the expectation that FeSOD and Fe2-MnSOD should ex- hibit identical absorption spectra and this too is contravened by the results reported herein. In a reciprocal observation, Yamakura (48) prepared the Mn-substituted FeSOD from Pseudomonas ovalis, and we notice that its spectrum differs from that of an authentic MnSOD. The complete specificity for metal, with regard to enzymatic activity, and the differ- ences in absorption spectra, lead to the view that the iron- binding site of FeSODs must differ from the manganese- binding site of MnSODs. This does not apply to the SOD from Bacterioides species, which are active with either iron or manganese at the active site (16-19).

Goodin (49) applied EXAFS and EPR to the MnSOD from T. thermophilus, both in the native state and after replace- ment of Mn by Fe. Chemical reduction of the Mn(II1) to Mn(1I) in the enzyme shifted the energy of the K-edge, but did not otherwise change the EXAFS spectrum. This indicates that reduction of the active site metal was not associated with rearrangement of the ligands. The Mn(II1) enzyme was, as expected, EPR silent, while the EPR spectrum of the Mn(I1) enzyme indicated axial symmetry. Combining this with the EXAFS data suggests an axial symmetry also for the Mn(II1) enzyme. The EPR spectrum of the Fe-substituted form of this MnSOD again indicated axial symmetry. This contrasts with the rhombic distortion deduced from the EPR spectra of all the native FeSODs thus far examined (3, 47, 50, 51). Goodin (49) also noted the similarity of the EPR spectra of the Fe- substituted MnSOD from T. thermophilus and the H20z- inactivated FeSOD from Propionibacterium sherrnanii (52). This recalls the similarity between the optical spectra of the Fez-MnSOD and the H20z-inactivated FeSOD of E. coli.

The x-ray crystallographic studies, which led to the view that the active sites of MnSODs and FeSODs are very similar, were based upon comparisons of SODs from different bacteria (8-12). The E. coli FeSOD has been studied by x-ray crystal- lography (5, 12), but the E. coli MnSOD has not yet been so studied. These two SODs exhibit 40% sequence identity (53) and gross conformational similarity is to be expected. Never- theless, their differences in metal specificity (14,23), chemical reactivities (14,54), and dissociation tendencies (26) indicate differences no: yet explainable in structural terms. High res- olution (-1.8A) x-ray diffraction studies of these two E. coli enzymes may be required to provide explanations for their behavioral differences.

1.

2.

3. 4.

5.

6. 7. 8.

10. 9.

11.

12.

13.

14. 15.

16.

17. 18. 19.

20.

21.

22.

23. 24.

25. 26.

27. 28. 29. 30. 31. 32. 33.

34. 35.

36. 37. 38. 39.

41. 40.

42. 43.

44.

45. 46.

47.

48. 49.

51. 50.

52.

53. 54.

REFERENCES DiGuiseppi, J., and Fridovich, I. (1984) CRC Crit. Reu. Toxicol. 12 , 315-

Keele, B. B., Jr., McCord, J. M., and Fridovich, I. (1970) J. Biol. Chem.

Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 2 4 8 , 4905-4908 Schinina, M. E., Maffey, L., Barra, D., Bossa, F., Puget, K., and Michelson,

A. M. (1987) FEES. Lett. 22 1,87-90 Carlioz, A,, Ludwig, M. L., Stallings, W. C., Fee, J. A,, Steinman, H. M.,

and Touati, D. (1988) J. Biol. Chem. 263 , 1555-1562 Steinman, H. M. (1978) J. Biol. Chem. 253,8708-8720 Takeda, Y., and Avila, H. (1986) Nucleic Acid Res. 11,4577-4587 Stallings, W. C., Pattridge, K. A,, Strong, R. K., and Ludwig, M. L. (1985)

Parker, M. W., and Blake, C. C. F. (1988) J. Mol. Biol. 199,649-661 Ringe, D., Petsko, G. A., Yamakura, F., Suzuki, K., and Ohmori, P. (1983)

Stallings, W. C., Powers, T. B., Pattridge, K. A., Fee, J. A., and Ludwig,

Stallings, W. C., Pattridge, K. A., Strong, R. K., and Ludwig, M. L. (1984)

Chan, V. W. F., Mjerrurn, M. J., and Borders, C. L. (1990) Arch. Biochem.

Ose, D. E., and Fridovich, I. (1976) J. Biol. Chem. 251,1217-1218 Clare, D. A., Blum, J., and Fridovich, I. (1984) J. Biol. Chem. 259 , 5932-

Gregory, E. M., and Dapper, C. H. (1983) Arch. Biochem. Biophys. 220 ,

Gregory, E. M. (1985) Arch. Biochem. Biophys. 238.83-89

Amano, A,, Shizukuishi, S., Tamagawa, H., Iwakura, S., Tsunasawa, S., Pennington, C. D., and Gregory, E. M. (1986) J. Bacteriol. 166,528-532

and Tsunemitsu, A. (1990) J. Bacteriol. 172, 1457-1463 Meier, B., Barra, D., Bossa, F., Calahrese, L., and Rotilio, G. (1982) J. Biol.

Chem. 2 5 7 , 13977-13980 Martin, M. E., Strachan, R. C., Aranha, H., Evans, S. L., Salin, M. L.,

Welch, B., Arceneaux, J. E. L., and Byers, B. R. (1984) J. Bacteriol. 159 , 745-749

342

245,6176-6181

J. Biol. Chem. 260,16424-16432

Proc. Natl. Acad. Sci. U. S. A . 8 0 , 3879-3883

M. L. (1983) Proc. Natl. Acad. Sci. U. S. A . 80,3884-3888

J. Biol. Chem. 2 5 9 , 10695-10699

Biophys. 279,195-201

5936

293-300

Martin, M. E., Byers, B. R., Olson, M. 0. J., Salin, M. L., Arceneaux, J. E.

Ose, D. E., and Fridovich, I. (1979) Arch. Biochem. Eiopfiys. 194,360-364 Kelle, B. B., Gioragnoli, C., and Rotilio, G. (1975) Physzol. Chem. Phys. 7 ,

. ~. . ._

L., and Tolhert, C. (1986) J. Biol. Chem. 261,9361-9367

1-6 Lawrence, G. D., and Sawyer, D. T. (1979) Biochemistry 18,3045-3050 Beyer, W. F., Jr., Reynolds, J. A,, and Fridovich, I. (1989) Biochemistry

Bjerrum, M. J. (1987) Biochim. Biophys. Acta 915,225-237 Beyer, W. F., Jr., and Fridovich, I. (1987) Biochemistry 2 6 , 1251-1257 Moody, C. S., and Hassan, H. M. (1984) J. Biol. Chem. 2 5 9 , 12821-12825 Hassan, H. M., and Moody, C. S. (1987) J. Biol. Chem. 262,17173-17177 Pugh, S. Y. R., and Fridovich, 1. (1985) J. Bacteriol. 162,196-202 Privalle, C. T., and Fridovich, I. (1988) J. Biol. Chem. 2 6 3 , 4274-4279 Privalle, C. T., Beyer, W. F., Jr., and Fridovich, I. (1989) J. Biol. Chem.

Britton, L., and Fridovich, I. (1978) Arch. Biochem. Eiophys. 191 , 198-205 Yang, J. T., Wu, C. C., and Martinez, H. M. (1986) Methods Enzymol. 130,

Ozols, J. (1990) Methods Enzymol. 182 , 587-601 McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244,6049-6055 Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427

Beauchamp, C. O., and Fridovich, I. (1971) Anal. Biochem. 44,276-287 Laemmli, U. K. (1970) Nature 227,680-685

Matsudaira, P. (1987) J. Biol. Chem. 262 , 10035-10038 Legendre, N., and Matsudaira, P. (1988) Biotechniqws 6 , 154-159 Chen, Y. H., and Yang, J. T. (1971) Biochem. Biophys. Res. Commun. 4 4 ,

Gel Filtration Theorv and Practice. Pharmacia Lahoratorv Separation Di-

28,4403-4409

264,2758-2763

208-209

1285-1291

vision, p. 32 "

Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182 , 319-326 McCord, J. M., Boyle, J., Day, E. D., Rizzolo, L. J., and Salin, M. L. (1977)

in Superoxide and Superoxide Dismutases (McCord, J. M., and Fridovich, I., eds) pp. 129-138, Academic Press, London

Dooley, D. M., Karas, J. L., Jones, T. F., Coti, C. E., and Smith, S. B. (1986) Inorg. Chem. 25,4761-4766

Yamakura, F., and Suzuki, K. (1978) J. Biochem. (Tokyo) 8 8 , 191-196 Goodin, D. B. (1983) Ph.D. dissertation, University of California, Berkeley

Villafranca, J. J. (1976) FEES Lett. 62, 230-232 Slykhouse, T. O., and Fee, J. A. (1976) J. Biol. Chem. 251,5472-5477

Twilfer, H., Gersonde, K., Meier, B., and Schwartz, A. L. (1980) in Chemical and Biochemical Aspects of Superoxide and Superoxide Dlsmutase (Ban- nister, J. V., and Hill, H. A. D., eds) pp. 168-175, Elsevier Science Publisher, Holland

Parker, M. W., and Blake, C. C. F. (1988) FEES Lett. 229,377-382 Borders, C. L., Jr., Horton, P. J., and Beyer, W. F., Jr. (1984) Arch.

Biochem. Biophys. 2 6 8 , 74-80