Probing structure–function relationships in early eventsin photosynthesis using a chimeric photocomplexKenji V. P. Nagashimaa,1, Mai Sasakib, Kanako Hashimotoc, Shinichi Takaichid,2, Sakiko Nagashimae,f, Long-Jiang Yug,Yuto Abeb, Kenta Gotoub, Tomoaki Kawakamib, Mizuki Takenouchib, Yuuta Shibuyab, Akira Yamaguchib,Takashi Ohnoc, Jian-Ren Sheng, Kazuhito Inouea,f, Michael T. Madiganh, Yukihiro Kimurac,1,and Zheng-Yu Wang-Otomob,1

aResearch Institute for Photobiological Hydrogen Production, Kanagawa University, Kanagawa 259-1293, Japan; bFaculty of Science and Institute of QuantumBeam Science, Ibaraki University, Mito 310-8512, Japan; cDepartment of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe657-8501, Japan; dDepartment of Biology, Nippon Medical School, Tokyo 180-0023, Japan; eDepartment of Biological Sciences, Tokyo Metropolitan University,Tokyo 192-0397, Japan; fResearch Institute for Integrated Science, Kanagawa University, Kanagawa 259-1293, Japan; gResearch Institute for InterdisciplinaryScience, Okayama University, Okayama 700-8530, Japan; and hDepartment of Microbiology, Southern Illinois University, Carbondale, IL 62901

Edited by Susan S. Golden, University of California, San Diego, La Jolla, CA, and approved August 29, 2017 (received for review March 3, 2017)

The native core light-harvesting complex (LH1) from the thermophilicpurple phototrophic bacterium Thermochromatium tepidum requiresCa2+ for its thermal stability and characteristic absorption maximumat 915 nm. To explore the role of specific amino acid residues of theLH1 polypeptides in Ca-binding behavior, we constructed a geneticsystem for heterologously expressing the Tch. tepidum LH1 complexin an engineered Rhodobacter sphaeroides mutant strain. This sys-tem contained a chimeric pufBALM gene cluster (pufBA from Tch.tepidum and pufLM from Rba. sphaeroides) and was subsequentlydeployed for introducing site-directed mutations on the LH1 poly-peptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-typeLH1 complex was isolated in a reaction center (RC)-associated formand displayed the characteristic absorption properties of this ther-mophilic phototroph. Spheroidene (the major carotenoid in Rba.sphaeroides) was incorporated into the Tch. tepidum LH1 complexin place of its native spirilloxanthins with one carotenoid moleculepresent per αβ-subunit. The hybrid LH1-RC complexes expressed inRba. sphaeroides were characterized using absorption, fluorescenceexcitation, and resonance Raman spectroscopy. Site-specific muta-genesis combined with spectroscopic measurements revealed thatα-D49, β-L46, and a deletion at position 43 of the α-polypeptide playcritical roles in Ca binding in the Tch. tepidum LH1 complex; in con-trast, α-N50 does not participate in Ca2+ coordination. These findingsbuild on recent structural data obtained from a high-resolution crys-tallographic structure of the membrane integrated Tch. tepidumLH1-RC complex and have unambiguously identified the locationof Ca2+ within this key antenna complex.

photosynthesis | light harvesting | Ca binding | Qy transition |Thermochromatium tepidum

Structural and functional research on bacterial photosynthesishas paved the way for our understanding of the basic mecha-

nism of solar energy conversion in natural systems. The early eventsin photosynthesis are carried out by two distinct components: theantenna apparatus for light energy collection and the reactioncenter (RC) for charge separation. In purple phototrophic bacteria,the antenna system consists of two large pigment-protein com-plexes: the core light-harvesting complex (LH1) that surrounds theRC and the peripheral light-harvesting complex (LH2) that sur-rounds the LH1. Both LH1 and LH2 are large oligomers formedfrom a basic structural subunit composed of two integral membranepolypeptides (α and β) that associate with bacteriochlorophyll(BChl) and carotenoid molecules. LH1 is present in all purplebacteria, whereas LH2 is absent in some species (1). In addition tolight harvesting, LH1 is also involved in quinone transport betweenthe RC and the quinone pool in the photosynthetic membrane (2).The LH1 complex from the thermophilic purple sulfur bacte-

rium Thermochromatium tepidum, first isolated from a Yellowstone

hot spring by Madigan (3), is unique in its characteristic red-shiftedQy absorption band at 915 nm (4, 5). This contrasts with theLH1 complexes from most other purple bacteria, which show Qyabsorbance of 875–890 nm. The Tch. tepidum LH1-RC corecomplex thus provides a unique model for studying “uphill” energytransfer from LH1 to the RC (6, 7). The unusual red shift of theTch. tepidum LH1-Qy has been attributed to the binding of Ca2+ tothe C-terminal domain of LH1 polypeptides (8, 9). A recentcrystallographic structure of the Tch. tepidum LH1-RC revealedthat LH1 forms concentric cylinders around the RC with the innerα- and outer β-polypeptide rings tightly connected by 16 Ca2+ onthe periplasmic membrane surface (10). The 3.0-Å electron densitymap suggested that each Ca2+ in LH1 was coordinated by the mainchain oxygen atom of α-Trp46, the side chains of α-Asp49 andα-Asn50, and the C-terminal carboxyl group of β-Leu46 in theadjacent subunit (10) (SI Appendix, Fig. S1). However, unambig-uous positioning of Ca2+ within the Tch. tepidum LH1-RC couldnot be deduced from the crystal structure alone.

Significance

Phototrophic bacteria have provided fundamental insight intothe biological mechanism of solar energy conversion. Early eventsin photosynthesis are carried out by the antenna apparatus forlight-harvesting (LH) and the reaction center (RC) for chargeseparation. Here we describe a system for expressing a chimericLH1-RC complex from two phylogenetically distant phototrophicpurple bacteria: the LH1 from Thermochromatium tepidum andthe RC from Rhodobacter sphaeroides. This system is exploitedto definitively localize Ca2+ within the Tch. tepidum LH1 com-plex. The hybrid photocomplexes also provide powerful newtools for probing photosynthetic energy transfer, identifyingintrinsic LH1–RC interactions, monitoring altered behavior ofcarotenoids in a nonnative environment, and linking specificamino acid residues to the specific spectroscopic properties ofdifferent phototrophic organisms.

Author contributions: K.V.P.N., Y.K., and Z.-Y.W.-O. designed research; K.V.P.N., M.S.,K.H., S.T., S.N., Y.A., K.G., T.K., M.T., Y.S., Y.K., and Z.-Y.W.-O. performed research;L.-J.Y., Y.S., A.Y., T.O., J.-R.S., K.I., and M.T.M. contributed new reagents/analytic tools;K.V.P.N., M.S., K.H., S.T., S.N., L.-J.Y., Y.A., K.G., T.K., M.T., A.Y., T.O., J.-R.S., K.I., M.T.M.,Y.K., and Z.-Y.W.-O. analyzed data; and K.V.P.N., M.T.M., Y.K., and Z.-Y.W.-O. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: wt503649bw@kanagawa-u.ac.jp,ykimura@people.kobe-u.ac.jp, or wang@ml.ibaraki.ac.jp.

2Present address: Department of Molecular Microbiology, Faculty of Life Sciences, TokyoUniversity of Agriculture, Tokyo 156-8502, Japan.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1703584114/-/DCSupplemental.

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To positively identify the Ca2+ coordinating residues and toreveal their contributions to the LH1-Qy shift, we constructed agenetic system for heterologously expressing the Tch. tepidumLH1 complex in a deletion strain of Rhodobacter sphaeroides, aphylogenetically distant relative of Tch. tepidum that is readilyamenable to genetic manipulation. By exploiting this system, wefunctionally expressed the Tch. tepidum LH1 complex in Rba.sphaeroides despite the two phototrophs sharing <40% sequenceidentity in their LH1 α- and β-polypeptides. In addition, thecarotenoid composition of Tch. tepidum LH1 was replaced withthat of Rba. sphaeroides. Site-directed mutants obtained fromthese constructions were then used to rigorously characterize theTch. tepidum LH1 complex and unequivocally identify how Ca2+,a cation that influences both the thermal stability and uniquespectral properties of Tch. tepidum LH1 (8, 11), is positionedwithin the precise structure of this important photocomplex.Although this system is shown to verify the Ca-binding site withinthe Tch. tepidum LH1 complex, the hybrid photocomplexes alsoprovide powerful new research tools for probing photosyntheticenergy transfer and quinone transport pathway, identifying in-trinsic LH1–RC interactions, monitoring altered behavior ofcarotenoid molecules in a nonnative environment, linking spe-cific amino acid residues to the specific spectroscopic propertiesof different phototrophic organisms, and advancing artificialphotosynthesis.

ResultsConstruction of a Heterologous Expression System for Synthesizingthe Tch. tepidum LH1 Complex and Production of Site-Directed Mutants.A mutant of Rba. sphaeroides strain IL106 lacking the RC, LH1,and LH2 complexes (designated strain DPP1) was used toconstruct a genetic system for synthesizing the Tch. tepidumLH1 polypeptides (12) (SI Appendix, Materials and Methods).The constructed strain (designated strain TS2) contains a chi-meric pufBALM gene cluster with pufBA from Tch. tepidum andpufLM from Rba. sphaeroides. Using this strain, site-specificmutations in Tch. tepidum LH1 were designed with the fol-lowing rationale. An alanine residue was inserted at position43 of the Tch. tepidum α-polypeptide (SI Appendix, Fig. S1),designated α-ins43A, to mimic the sequence of the LH1 com-plex (Qy at 889 nm) of Allochromatium vinosum, a mesophilicrelative of Tch. tepidum, and to examine the effect of this de-letion on the LH1-Qy transition. The amino acids α-Asp49,α-Asn50, and β-Leu46 in Tch. tepidum LH1 were replaced byalanines (designated α-D49A, α-N50A, and β-L46A, respec-tively), because these residues had been tentatively assigned asCa-coordinating ligands in the LH1-RC crystal structure (10).Finally, to specifically assess the role of the C-terminal residueof the β-polypeptide in Ca2+ binding, a deletion mutant of theβ-Leu46, designated β-L46del, was prepared.

Expression and Properties of the Chimeric LH1-RC Complexes. AllRba. sphaeroides strains harboring the chimeric pufBALM geneclusters grew phototrophically at virtually similar growth ratesas Rba. sphaeroides (SI Appendix, Fig. S2). The heterologouslyexpressed Tch. tepidum LH1 polypeptides were assembled asfunctional pigment-protein complexes in all strains, as confirmedby the absorption spectra of intact cells and intracytoplasmicmembrane (ICM) preparations (Fig. 1 and SI Appendix, Fig. S3).The identities of the expressed products were confirmed by TOF/MS measurements. No PufX polypeptide was detected in thechimeric LH1-RCs from both TOF/MS and HPLC analyses.The LH1 complex from Rba. sphaeroides strain TS2 contained thewild-type sequences of Tch. tepidum and showed a Qy transitionat ∼915 nm identical to that of the native LH1 complex fromTch. tepidum (13). This result demonstrates that Rba. sphaeroidescan effectively express LH1 complexes from a purple bacteriumwith significantly different LH1 polypeptides and spectroscopic

properties from those of the native Rba. sphaeroides LH1. It alsoimplies that the LH1-Qy position is primarily defined by thestructures of the α- and β-polypeptides.The LH1 polypeptides in strains TS2 and α-N50A were well

assembled with BChl a, whereas mutant strains α-43insA, α-D49A,β-L46A, and β-L46del contained variable levels of BChl a thatwere not properly incorporated within the LH1 polypeptides, asshown by their absorption characteristics between 750 and 800 nm(SI Appendix, Fig. S3). There was a tendency for strains expressingLH1 complexes with Qy bands more blue-shifted from that of thenative Tch. tepidum LH1 to have higher proportions of mis-assembled complexes; this implies that the site-specific mutationsthat induce larger structural changes have more detrimental ef-fects on pigment assembly. However, most of the misassembledcomponents could be removed from the crude ICMs by milddetergent-treatment (Fig. 1). Unless stated otherwise, the detergent-treated ICMs of α-43insA, α-D49A, β-L46A, and β-L46del wereused in subsequent measurements.Introduction of different site-specific mutations into the Tch.

tepidum LH1 polypeptides resulted in LH1 complexes with dif-fering Qy transitions (Fig. 1). The LH1 complex of strain α-N50Ashowed a Qy at 915 nm, the same as that of the native Tch.tepidum LH1. This indicates that the asparagine residue, the 50thresidue from the N terminus in the α-polypeptide (Asn50), doesnot participate in Ca binding in the Tch. tepidum LH1 complex,in agreement with preliminary results from a 1.9-Å structure ofthe Tch. tepidum LH1-RC (14). In contrast, the LH1 from strainα-43insA, which contains an Alc. vinosum-like α-polypeptidesequence (15), exhibited a blue-shifted Qy band at 899 nm.Comparison of this with the native Alc. vinosum LH1-Qy(889 nm) (16) suggests that the deletion at position 43 in the Tch.tepidum α-polypeptide contributes significantly to the Qy redshift. Mutation of the α-Asp49 to Ala (strain α-D49A) resulted ina blue-shifted Qy band at 892 nm. This result agrees with theassignment of this residue as a Ca-binding ligand in the crystalstructure (10), and indicates that this residue also plays an

Fig. 1. Absorption spectra of crude (strains TS2 and α-N50A) and detergent-treated (strains α-43insA, α-D49A, β-L46del and β-L46A) ICMs from the Rba.sphaeroides mutant strains. All membrane preparations were suspended in20 mM Tris·HCl (pH 8.5) buffer and the spectra were recorded at roomtemperature.

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important role in maintaining the red-shifted Qy of the Tch.tepidum LH1 complex. Both deletion and substitution of theβ-Leu46 residue resulted in blue-shifted Qy transitions, high-lighting the importance of this C-terminal residue. In fact, theβ-L46A substitution yielded the most blue-shifted Qy transitionof all mutations introduced in this study (Fig. 1).

Spectroscopic Characterization of the Strain TS2 Chimeric LH1-RCComplex. The LH1 complex from strain TS2 (Fig. 1) was suffi-ciently stable to be solubilized in an LH1-RC form and wasobtained at high purity. Fig. 2 compares the spectra of purifiedLH1-RC complexes from strains TS2, DP2 (an LH2-deficient Rba.sphaeroides strain; SI Appendix, Materials and Methods), and nativeTch. tepidum. Typically, the chimeric LH1-RC from strain TS2displayed an LH1-Qy band in the range of 915–917 nm. A re-markable feature is the incorporation of spheroidene (the majorcarotenoid in Rba. sphaeroides) into the LH1 complex of strainTS2 in place of spirilloxanthin, which is present in the native Tch.tepidum LH1 complex (13). Notably, however, fewer spheroidenesper LH1 complex were incorporated than in wild-type Rba.sphaeroides LH1. Quantitative carotenoid analyses revealed thepresence of 17.6 ± 1.0 carotenoid molecules per LH1-RC of strainTS2, indicating that each LH1 αβ pair incorporated only one ca-rotenoid molecule. This contrasts with the two carotenoid mole-cules per αβ pair in the LH1 complex from strain DP2, the same asin wild-type Rba. sphaeroides (17).Table 1 shows the carotenoid composition of purified LH1-RC

complexes from the Rba. sphaeroides expression system. Spher-oidene was the major component in all complexes, although therewere variations in the composition of spheroidene derivativesamong the complexes. The spheroidene-dominant carotenoids inthe LH1 complex of strain TS2 exhibited absorption maxima at443, 470, and 503 nm, slightly blue-shifted from those of thecarotenoids in the LH1 complex of strain DP2 (447, 472, and505 nm). This may reflect a difference in the protein environmentbetween the two LH1 complexes (18).The existence of Ca-binding sites in the LH1 from strain TS2

was probed using EDTA titration experiments. Fig. 2, Inset showsa typical example in which on addition of EDTA, the LH1-Qyband blue-shifted from 915 nm to 895 nm and displayed abroadened band shape. The absorption maximum then red-shifted

back to 912 nm following the addition of excess CaCl2. These dataprovide additional evidence that the Ca-binding sites in Tchtepidum LH1 are also present in the heterologously expressedLH1 complex.Fig. 3 compares the steady-state fluorescence excitation spectrum

of the purified LH1-RC complex from strain TS2 with the spectrafrom strain DP2 and from Tch. tepidum. The overall efficiency ofexcitation energy transfer from the nonnative carotenoids to BChl ain the LH1 complex from strain TS2 was calculated as 44%. Thisvalue is significantly lower than that of the native Rba. sphaeroidesLH1 complex from strain DP2 (71%) despite a similar carotenoidcomposition, but is much higher than that of the spirilloxanthin-containing Tch. tepidum LH1 complex (23%). This result suggeststhat energy transfer efficiency may be affected not only by theconjugation length of the carotenoid, but also by the protein envi-ronment, as well as the gap between the absorption maxima of thecarotenoid and BChl a molecules and the number of carotenoidspresent in the LH1 complex. The efficiencies for the LH1 com-plexes from strain DP2 and native Tch. tepidum are compatible withthose reported for other spheroidene- and spirilloxanthin-containingLH1 complexes, respectively (19–22).The characteristics of chimeric and native LH1-RC complexes

were further examined by resonance Raman spectroscopy. Onexcitation at 532 nm in the absorption region of carotenoids, theLH1 from strain TS2 yielded a spectrum essentially identical tothat from strain DP2 (Fig. 4B), a spectrum characteristic of all-trans spheroidene. The intense bands at 1,518 cm−1 and 1,154 cm−1,assigned to the νC=C and νC-C/δCH modes of all-trans spher-oidene, respectively (23), were shifted by 16 cm−1 and 8 cm−1 fromthose of all-trans spirilloxanthin in Tch. tepidum LH1 (24) dueto the relatively shorter CC bond lengths. The intense peak at1,518 cm−1 in strain TS2 LH1 contained a shoulder around1,505 cm−1, which became more apparent or split into two peaks inthe spectra of mutant LH1 complexes (Fig. 4).Interactions between BChl a and LH1 polypeptides were in-

vestigated by near-infrared resonance Raman spectroscopy (Fig.5). The C3-acetyl C=O stretching band of the TS2 LH1 at1,640 cm−1 was up-shifted by 3 cm−1 from that of native Tch.tepidum LH1 (24) but down-shifted 7 cm−1 from that of strainDP2 LH1. This result indicates that the interaction between theC3-acetyl group of BChl a and the LH1 polypeptides in strainTS2 is slightly weaker than that in the native Tch. tepidumLH1 complex but is stronger than that in the wild-type Rba.sphaeroides LH1 complex. As for the BChl a C13-keto group, theC=O stretching band at 1,676 cm−1 for strain TS2 LH1 is closeto that of wild-type Tch. tepidum LH1, but significantly blue-shifted by 10 cm−1 from that of strain DP2 LH1. These datasuggest a very weak (if any) hydrogen-bonding interaction be-tween the C13-keto group and the LH1 polypeptides of strainTS2, whereas such hydrogen bonds likely form in the strain DP2LH1 complex (25).

Characterization of Site-Specific Mutants of Tch. tepidum LH1Complexes. Because spheroidene was incorporated into the chi-meric LH1-RC complexes as the major carotenoid, the resonance

Fig. 2. Comparison of the absorption spectrum of the chimeric LH1-RCcomplex purified from strain TS2 (red curve) with the spectra of the LH1-RCcomplexes purified from strain DP2 (black curve) and from Tch. tepidum(dotted curve). All spectra were normalized at LH1-Qy bands. (Inset) Changesin the absorption spectra of the TS2 LH1-RC (red) on addition of EDTA to afinal concentration of 5 mM (blue curve), followed by addition of CaCl2 to afinal concentration of 175 mM (dashed curve).

Table 1. Carotenoid composition (mol % of total carotenoids) inpurified LH1-RC complexes from Rba. sphaeroides strains DP2,TS2, and α-N50A

Carotenoid DP2 TS2 α-N50A

OH-spheroidene 23.8 6.9 7.5Spirilloxanthin 0.9 7.7 6.3Demethylspheroidene 9.9 9.4 15.6Spheroidenone 3.0 1.8 1.1Spheroidene 62.4 74.2 69.5

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Raman spectra excited at 532 nm for the mutants were basicallyidentical to those of wild-type Rba. sphaeroides LH1-RC. However,distinct differences were observed for the LH1 mutants in the C=Cstretching region (Fig. 4), in which the intense bands around1,518 cm−1 split into two bands, one around 1,505–1,508 cm−1 andthe other around 1,516–1,519 cm−1. The relative intensity of theformer to the latter increased in the order of β-L46del > α-D49A >α-N50A > α-ins43A > TS2. These results likely reflect a distortedconformation of the spheroidene molecules from a planar geom-etry along the C=C backbone in the mutant LH1 complexes (26).If this is so, then such distortion could influence the properties ofBChl a in the LH1 complex and can be detected by resonanceRaman spectroscopy with excitation at 1,064 nm. The resultspresented in Fig. 5 show that α-N50A with its LH1-Qy band at915 nm gave a C3-acetyl C=O stretching band at 1,640 cm−1,whereas this band shifted to 1,642 cm−1 for α-ins43A and β-L46deland to 1,647 cm−1 for α-D49A.A linear relationship exists between a downshift in the C3-acetyl

stretching mode, which is sensitive to hydrogen bonding, and a redshift in the LH Qy maximum (25, 27) (SI Appendix, Fig. S4). Thisindicates that hydrogen-bonding interactions indeed occur in theLH1 complexes of the chimeric mutants. Moreover, the ratio of thesplits in the carotenoid νC=C bands (∼1,505 cm−1/∼1,519 cm−1)also reveal a correlation with the LH1-Qy red shift. This findingindicates that conformational changes of carotenoid molecules

have little if any effect on LH1 absorption properties, but do in-fluence the LH1-Qy transition energy.

DiscussionThe Tch. tepidum LH1 complex has been successfully expressedin Rba. sphaeroides mutant strains. In addition to their use inprobing the coordination and function of Ca in Tch. tepidumLH1, these strains are valuable new tools for exploring severalkey primary processes in photosynthesis in greater detail. Theseinclude, but are not limited to (i) producing various hybrid LH1-RC complexes with vastly different biochemical and spectro-scopic properties; (ii) investigating the intrinsic nature of theLH1–RC interactions; and (iii) verifying the roles of specificamino acids in energy transfer-coupled electron/proton transportprocesses. Because the LH1 complex surrounds the RC, it servesas both the entrance route of excitation energy to the RC and theexit route of reduced ubiquinones from the RC to the quinonepool and cytochrome bc1 complex (2). Therefore, the chimericLH1-RC complexes constructed herein also provide excellentsubjects for probing the dynamic processes of energy and elec-tron transfers in photosynthesis by time-resolved spectroscopy.At present, no genetic manipulation or expression system is

available for Tch. tepidum, while several heterologous expressionsystems exist in other species and have been used to study variousfunctions of the bacterial photosynthetic apparatus (28–33).However, our construction differs from other chimeric LH1-RCsin at least three major ways: (i) the complex contains compo-nents from distantly related purple bacteria, the purple sulfurbacterium Tch. tepidum (γ-Proteobacteria) and the purple non-sulfur bacterium Rba. sphaeroides (α-Proteobacteria); (ii) theLH1 transferred to Rba. sphaeroides differs significantly in pri-mary structure from its native LH1 complex (37% sequenceidentity for both α- and β-polypeptides); and (iii) the Qy transi-tion of the chimeric LH1 absorbs light of significantly longer

A

B

C

D

E

F

G

Fig. 4. Resonance Raman spectra with excitation at 532 nm for the purifiedLH1-RC complexes from Tch. tepidum (A), strain DP2 (B), and ICM fromstrains TS2 (C), α-43insA (D), α-N50A (E), α-D49A (F), and β-L46del (G).

A

B

C

Fig. 3. Analysis of the efficiency of steady-state excitation energy transferfrom carotenoid to BChl a in purified LH1-RC complexes from strains DP2 (A)and TS2 (B), and from Tch. tepidum (C). Fluorescence emission spectra(dashed lines) were plotted to match the maximum intensities to those ofthe LH1-Qy bands in fractional absorption spectra (1 − T, where T is trans-mittance) (solid black) on the same scale. Fluorescence excitation spectra(red) detected at fluorescence emission maxima were normalized at LH1Soret bands in fractional absorption spectra.

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wavelengths compared with that of the RC in the host strain(915 nm vs. 868 nm for the special pair).All of the modified LH1 complexes produced in this study

supported anaerobic, phototrophic growth of Rba. sphaeroides,and similar growth rates as seen in strain DP2 were observed forall mutant strains. These findings indicate that light energyabsorbed by the modified LH1 complexes is indeed transferredto the RC for charge separation, even though these are ener-getically “uphill” processes (∼490 cm−1) occurring within astructurally nonnative LH1-RC complex. Phototrophic growthalso demonstrates that the electron and proton transports nec-essary to support photophosphorylation are operational in eachmutant derivative. Thus, in addition to the applications listedpreviously, the chimeric photocomplexes described here makeavailable a powerful platform for experimentally dissecting themechanism of uphill energy transfer and related early eventsin photosynthesis.The characteristic feature of the LH1-Qy transition in native

Tch. tepidum was reproduced in the heterologously expressed LH1complex containing wild-type sequences, indicating that thespectroscopic properties of BChl a molecules in LH1 complexesare ultimately determined by the primary structures of α- andβ-polypeptides. As revealed by resonance Raman spectroscopy(Fig. 5), the interaction between BChl a and LH1 polypeptides inthe heterologously expressed LH1 complex containing wild-typesequences (strain TS2) was virtually identical to that seen in thenative Tch. tepidum LH1 complex. The near-infrared Raman re-sults show that the hydrogen-bonding interactions between theBChl a C3-acetyl group and LH1 polypeptides in the native Tch.tepidum (24) are also major factors regulating the Qy transition ofthe expressed LH1 complexes.

The PufX polypeptide in Rba. sphaeroides is known to haveimportant roles in supporting photosynthetic growth, structuralorganization of the LH1-RC core complex, and cyclic electrontransfer (34). In this work, the Rba. sphaeroides pufX gene waspresent and unaltered in the mutant derivatives (SI Appendix,Fig. S5); however, its expression product could not be detected inthe chimeric LH1-RC complexes. While we are still attemptingto detect PufX in the chimeric complexes, our results thus farsuggest that the PufX polypeptide, if expressed, is not requiredfor (or is unable to promote) assembly of the Tch. tepidum LH1in Rba. sphaeroides, and thus is not incorporated in the LH1-RC complex.Site-specific mutagenesis of this study provided an ideal tool for

clarifying uncertainties in the assignments of the crystallographicelectron density map for the Ca-binding residues in the Tch. tep-idum LH1 complex. Both α-Asp49 and α-Asn50 were tentativelyassigned as the Ca-binding residues in the 3.0-Å structure (10).Mutation of the α-Asp49 to Ala resulted in a blue shift of the LH1-Qy transition to 892 nm, whereas mutation of the α-Asn50 to Aladid not alter the Qy position. These results are completely con-sistent with recent findings from both high-resolution Ca-bound(14) and Sr(Ba)-substituted Tch. tepidum LH1-RC structures (35)that point to α-Asp49 as a key metal-binding residue; in contrast, itis now clear that α-Asn50 does not participate in Ca coordination.In addition, our results for the two β-L46 mutants highlight theimportance and sensitivity of this C-terminal residue. Substitutionof the C-terminal β-Leu46 residue resulted in a blue-shifted LH1-Qy transition (Fig. 1). This result would be difficult to interpret ifthe C-terminal carboxyl group serves as a ligand to Ca, as has beententatively assigned in the 3.0-Å structure (10). Recently, it hasbecome clear from a high-resolution structure of the Tch. tepidumLH1-RC (14) that β-Leu46 is not directly involved in Ca binding,but does participate in an extensive hydrogen-bonding networkwith its neighboring β-polypeptide and water molecules around theC-terminal end. Deletion of β-Leu46 also resulted in a blue-shiftedLH1-Qy band to 897 nm, which can be attributed to a disruption ofthe hydrogen-bonding network and a conformational change in theC-terminal region of the β-polypeptide.The major difference between the native and heterologously

expressed Tch. tepidum LH1 complexes was their carotenoidcompositions. As spheroidene was biosynthetically incorporatedinto the expressed LH1 complexes in place of the native spi-rilloxanthin, our heterologous LH1 expression system could alsoserve as a powerful tool for probing the behavior of differentcarotenoids in a nonnative environment, as evident from theresonance Raman results (Fig. 4). Splittings of the νC=C bandsin the 1,500–1,520 cm−1 region were observed for carotenoids inthe expressed LH1 complexes, indicating either that some of thespheroidenes in the expressed LH1 complexes differ slightly intheir conformations from those in the native Rba. sphaeroidesLH1 complex or that minor carotenoids such as demethyl-spheroidene and/or spirilloxanthin may contribute to the low-frequency component (SI Appendix, Results and Discussion).A final benefit of our use of site-directed mutagenesis in this

study is that it allowed direct verification of a long-standing hy-pothesis on the effect of a deletion in the Tch. tepidum LH1α-polypeptide (8, 9, 11). Despite the high degree of sequenceidentities between Tch. tepidum and Alc. vinosum LH1 poly-peptides (SI Appendix, Fig. S1), a deletion is present at position43 in the Tch. tepidum LH1 α-polypeptide where an Ala exists inthe corresponding Alc. vinosum LH1 polypeptide (15). Insertionof an Ala residue into the Tch. tepidum LH1 α-polypeptide atthis position (strain α-43insA) resulted in a blue shift of its LH1-Qy band to 899 nm (Fig. 1). This indicates that the inserted Alain the Tch. tepidum LH1 α-polypeptide prevents formation of aproper Ca-binding pocket and subsequent incorporation of Ca2+

into the complex; this is not required in the mesophilic Alc.

A

B

C

D

E

F

G

Fig. 5. Near-infrared resonance Raman spectra with excitation at 1,064 nmfor the purified LH1-RC complexes from Tch. tepidum (A) and strain DP2(B), and ICM from strains TS2 (C), α-43insA (D), α-N50A (E), α-D49A (F), andβ-L46del (G).

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vinosum, since Ca2+ has no apparent effect on the spectroscopicproperties of its LH1-RC complex (36).

Materials and MethodsConstruction of a Rba. sphaeroidesmutant lacking genes encoding the LH1-RC(pufBALM) and LH2 (pucBA) polypeptides (designated DPP1) was performedusing Rba. sphaeroides strain IL106 (12). A mutant lacking only LH2, desig-nated DP2, was generated as well. The chimeric puf operon consisting of Tch.tepidum pufBA and Rba. sphaeroides pufLMXwas cloned with a suicide vectorpJP5603 and introduced into the Rba. sphaeroides mutant strain DPP1 byconjugal transfer from Escherichia coli S17-1λpir. The mutant recoveringphototrophic growth by incorporation of the chimeric puf construct to thegenomic DNA through a homologous recombination was named TS2. Site-specific mutagenesis and other genetic manipulations on the Tch. tepidumLH1 polypeptides were performed as described in SI Appendix, Materials andMethods. All expression strains were grown under phototrophic (anoxic/whitelight) conditions and, for certain experiments, with 850-nm LED illumination.The LH1-RC complexes from strains DP2, TS2, and α-N50A were purified bysolubilizing the ICM with 0.23% lauryldimethylamine N-oxide in 20 mM

Tris·HCl (pH 8.5), followed by anion-exchange chromatography (Toyopearl650S; TOSOH) with 0.1% n-dodecyl β-D-maltopyranoside at 4 °C. Details ofsample preparation and spectroscopic measurements are provided in SI Ap-pendix, Materials and Methods.

ACKNOWLEDGMENTS. We thank Ayumi Imai and Megumi Kobayashi fortheir technical assistance in preparing the LH1-RC complex from strain DP2,and the Kao Corporation for providing lauryldimethylamine N-oxide. Thiswork was supported by the Ministry of Education, Culture, Sports, Science,and Technology of Japan (MEXT; Grants-in-Aid for Scientific Research B16H04174, to Z.-Y.W.-O. and C 16K07295, to Y.K.), Takeda Science Foundation,a Sumitomo Foundation Grant for Basic Science Research Projects, the KurataMemorial Hitachi Science and Technology Foundation (Z.-Y.W.-O.), and aJapan Society for the Promotion of Science Research (JSPS) Fellowship (toT.K.). This work was supported in part by Grant-in-Aid for JSPS KAKENHI Grant15K00642, MEXT KAKENHI Grant 24107004, and the Strategic Research BaseDevelopment Program for Private Universities from MEXT, Japan (to K.I.).K.V.P.N. was supported in part by grants from the Tokyo Ohka Foundationand the Precursory Research for Embryonic Science and Technology programof the Japan Science and Technology Agency.

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