Different mechanisms of the binding of soluble electron donors to the photosynthetic reaction center of Rubrivivax gelatinosus and Blastochloris viridis

The tetraheme cytochrome subunits of the photosynthetic reaction centers (RCs) in two species of purple bacteria, Rubrivivax gelatinosus and Blastochloris (Rhodopseudomonas) viridis, were compared in terms of their capabilities to bind different electron-donor proteins. The wild-type RCs from both species and mutated forms of R. gelatinosus RCs (with amino acid substitutions introduced to the binding domain for electron-donor proteins) were tested for their reactivity with soluble cytochromes and high potential iron-sulfur protein. Cytochromes from both species were good electron donors to the B. viridis RC and the R. gelatinosus RC. The reactivity in the R. gelatinosus RC showed a clear dependence on the polarity of the charges introduced to the binding domain, indicating the importance of the electrostatic interactions. In contrast, high potential iron-sulfur protein, presumed to operate according to the hydrophobic mechanism of binding, reacted significantly only with the R. gelatinosus RC. Evolutionary substitution of amino acids in a region of the binding domain on the cytochrome subunit surface probably caused the change in the principal mode of protein-protein interactions in the electron-transfer chains.     

Comparative analysis of the primary structure of the reaction center-bound cytochrome subunit in purple bacteria

The amino acid sequences of the reaction center-bound cytochrome subunit of six species of purple bacteria were compared. Amino acid residues thought to be important in controlling the redox midpoint potentials of four hemes in Blastochloris (Rhodopseudomonas) viridis were found to be well conserved. As opposed to all other species studied, the amino acid sequence of the cytochrome subunit of B. viridis had several insertions of more than 10 residues at specific regions close to the LM core, suggesting that interaction of the cytochrome subunit with the LM core in most species is different from that in B. viridis. Distribution of charged amino acid residues on the surface of the cytochrome subunit was compared among six species and discussed from the viewpoint of interaction with soluble electron donors.     

Mutational analyses of the photosynthetic reaction center-bound triheme cytochrome subunit and cytochrome c2 in the purple bacterium Rhodovulum sulfidophilum

The purple photosynthetic bacterium Rhodovulum sulfidophilum has an unusual reaction center- (RC-) bound cytochrome subunit with only three hemes, although the subunits of other purple bacteria have four hemes. To understand the electron-transfer pathway through this subunit, three mutants of R. sulfidophilum were constructed and characterized: one lacking the RC-bound cytochrome subunit, another one lacking cytochrome c(2), and another one lacking both of these. The mutant lacking the RC-bound cytochrome subunit was grown photosynthetically with about half the growth rate of the wild type, indicating that the presence of the cytochrome subunit, while not indispensable, is still advantageous for the photosynthetic electron transfer to support its growth. The mutant lacking both the cytochrome subunit and cytochrome c(2) showed a slower rate of growth by photosynthesis (about a fourth of that of the wild type), indicating that cytochrome c(2) is the dominant electron donor to the RC mutationally devoid of the cytochrome subunit. On the other hand, the mutant lacking only the cytochrome c(2) gene grew photosynthetically as fast as the wild type, indicating that cytochrome c(2) is not the predominant donor to the RC-bound triheme cytochrome subunit. We further show that newly isolated soluble cytochrome c-549 with a redox midpoint potential of +238 mV reduced the photooxidized cytochrome subunit in vitro, suggesting that c-549 mediates the cytochrome c(2)-independent electron transfer from the bc(1) complex to the RC-bound cytochrome subunit. These results indicate that the soluble components donating electrons to the RC-bound triheme cytochrome subunit are somewhat different from those of other purple bacteria.     

A new cytochrome subunit bound to the photosynthetic reaction center in the purple bacterium, Rhodovulum sulfidophilum

The nucleotide sequence of the puf operon, which contains the genes encoding the B870 light-harvesting protein and the reaction center complex of the purple photosynthetic bacterium, Rhodovulum sulfidophilum, was determined. The operon, which consisted of six genes, pufQ, pufB, pufA, pufL, pufM, and pufC, is a new variety in photosynthetic bacteria in the sense that pufQ and pufC coexist. The amino acid sequence of the cytochrome subunit of the reaction center deduced from the pufC sequence revealed that this cytochrome contains only three possible heme-binding motifs; the heme-1-binding motif of the corresponding tetraheme cytochrome subunits was not present. This is the first exception of the "tetraheme" cytochrome family in purple bacteria and green filamentous bacteria. The pufC sequence also revealed that the sixth axial ligands to heme-1 and heme-2 irons were not present in the cytochrome either. This cytochrome was actually detected in membrane preparation as a 43-kDa protein and shown to associate functionally with the photosynthetic reaction center as the immediate electron donor to the photo-oxidized special pair of bacteriochlorophyll. This new cytochrome should be useful for studies on the role of each heme in the cytochrome subunit of the bacterial reaction center and the evolution of proteins in photosynthetic electron transfer systems.     

Phylogenetic Distribution of Unusual Triheme to Tetraheme Cytochrome Subunit in the Reaction Center Complex of Purple Photosynthetic Bacteria

To understand the evolutionary relationship between triheme and tetraheme cytochrome subunits in the reaction center complex, genes located downstream of that coding for the M subunit of the reaction center complex (pufM) were amplified by PCR and analyzed in six established and two unidentified species of the genus Rhodovulum and five species of the genus Rhodobacter. All the Rhodovulum species tested had the pufC gene coding for the reaction-center-bound cytochrome subunit, while all the Rhodobacter species were found to have the pufX gene at the corresponding position. Analyses of the amino acid sequences of the pufC gene products showed that the cytochrome subunits of all the Rhodovulum species have three heme-binding-motifs and lack a methionine residue probably working as the sixth axial-ligand to one of the three hemes. Phylogenetic relationships among Rhodovulum species based on the pufC gene products were basically consistent with those based on 16S rRNA sequences, suggesting that the basic characteristics of the triheme cytochrome subunit have been conserved during the evolutionary process of the Rhodovulum species.     

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.     

Tuning heme redox potentials in the cytochrome C subunit of photosynthetic reaction centers

The photosynthetic reaction center (RC) from Rhodopseudomonas viridis contains four cytochrome c hemes. They establish the initial part of the electron transfer (ET) chain through the RC. Despite their chemical identity, their midpoint potentials cover an interval of 440 mV. The individual heme midpoint potentials determine the ET kinetics and are therefore tuned by specific interactions with the protein environment. Here, we use an electrostatic approach based on the solution of the linearized Poisson-Boltzmann equation to evaluate the determinants of individual heme redox potentials. Our calculated redox potentials agree within 25 meV with the experimentally measured values. The heme redox potentials are mainly governed by solvent accessibility of the hemes and propionic acids, by neutralization of the negative charges at the propionates through either protonation or formation of salt bridges, by interactions with other hemes, and to a lesser extent, with other titratable protein side chains. In contrast to earlier computations on this system, we used quantum chemically derived atomic charges, considered an equilibrium-distributed protonation pattern, and accounted for interdependencies of site-site interactions. We provide values for the working potentials of all hemes as a function of the solution redox potential, which are crucial for calculations of ET rates. We identify residues whose site-directed mutation might significantly influence ET processes in the cytochrome c part of the RC. Redox potentials measured on a previously generated mutant could be reproduced by calculations based on a model structure of the mutant generated from the wild type RC.     

Cooperative interaction of high-potential hemes in the cytochrome subunit of the photosynthetic reaction center of bacterium <Emphasis Type="Italic">Ectothiorhodospira shaposhnikovii</Emphasis>

Cooperative interaction of the high-potential hemes (C_h) in the cytochrome subunit of the photosynthesizing bacterium Ectothiorhodospira shaposhnikovii was studied by comparing redox titration curves of the hemes under the conditions of pulse photoactivation inducing single turnover of electron-transport chain and steady-state photoactivation, as well as by analysis of the kinetics of laser-induced oxidation of cytochromes by reaction center (RC). A mathematical model of the processes of electron transfer in cytochrome-containing RC was considered. Theoretical analysis revealed that the reduction of one heme C_h facilitated the reduction of the other heme, which was equivalent to a 60 mV positive shift of the midpoint potential. In addition, reduction of the second heme C_h caused a three-to four-fold acceleration of the electron transfer from the cytochrome subunit to RC. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 11, pp. 1540–1547.     

Structural and spectroscopic properties of a reaction center complex from the chlorosome-lacking filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii

The photochemical reaction center (RC) complex of Roseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemes c, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation from Chloroflexus aurantiacus. Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested that R. castenholzii had diverged from C. aurantiacus very early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. Although R. castenholzii is phylogenetically related to C. aurantiacus, the arrangement of its puf genes, which code for the light-harvesting proteins and the RC subunits, was different from that in C. aurantiacus and similar to that in purple bacteria. The genes are found in the order pufB, -A, -L, -M, and -C, with the pufL and pufM genes forming one continuous open reading frame. Since the photosynthetic apparatus and genes of R. castenholzii have intermediate characteristics between those of purple bacteria and C. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.     

In vitro and in vivo electron transfer to the triheme cytochrome subunit bound to the photosynthetic reaction center complex in the purple bacterium Rhodovulum sulfidophilum.

The cytochrome subunit bound to the photosynthetic reaction center (RC) complex in Rhodovulum sulfidophilum lacks one heme-binding motif (CXXCH) out of four motifs found in other purple bacteria resulting in the absence of the most distal heme from the RC-core complex (S. Masuda et al., J. Biol. Chem. 274 (1999) 10795). Cytochrome c(2), which acts as the electron donor to the RC was purified, and its gene was cloned and sequenced. The redox midpoint potential of cytochrome c(2) was determined to be E(m)=357 mV. The photo-oxidation and re-reduction of purified cytochrome c(2) were observed in the presence of membrane preparations. Flash-induced photo-oxidation and re-reduction of the RC-bound cytochrome were also observed in intact cells. Despite the unusual nature of the RC-bound cytochrome subunit, the cyclic electron transfer system in Rdv. sulfidophilum was shown to be similar to those in other purple bacteria.     

Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.

The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes.     

Structure of the H subunit of the photosynthetic reaction center from the thermophilic purple sulfur bacterium, Thermochromatium tepidum Implications for the specific binding of the lipid molecule to the membrane protein complex.

The photosynthetic reaction center (RC) is a transmembrane protein complex that catalyzes light-driven electron transport across the photosynthetic membrane. The complete amino-acid sequence of the H subunit of the RC from a thermophilic purple sulfur bacterium, Thermochromatium tepidum, has been determined for the first time among purple sulfur bacteria. The H subunit consists of 259 amino acids and has a molecular mass of 28 187. The deduced amino-acid sequences of this H subunit showed a significant (40%) degree of identity with those from mesophilic purple nonsulfur bacteria. The determined primary structure of the H subunit was compared with the structures of mesophilic B. viridis and R. sphaeroides based on the three-dimensional structure of the H subunit from T. tepidum, which has been recently determined by X-ray crystallography. One lipid molecule was found in the crystal structure of the T. tepidum RC, and the head group of the lipid appears to be stabilized by the electrostatic interactions with the conserved basic residues in the H subunit. The above comparison has suggested the existence of a lipid-binding site on the molecular surface at which a lipid molecule can interact with the RC in a specific manner.     

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.

The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.     

Electrostatic interaction between redox cofactors in photosynthetic reaction centers

Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921-3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them.     

Organization of the genes coding for the reaction-centre L and M subunits and B870 antenna polypeptides α and β from the aerobic photosynthetic bacterium Erythrobacter species OCH114

In the aerobic photosynthetic bacterium Erythrobacter species OCH114 the structural genes coding for the light-harvesting (LH) complex B870 and the reaction-centre (RC) polypeptides (the gene products of the pufB, pufA, pufL and pufM genes) are mapped on a 2.728 kbp EcoRI fragment. Sequencing of this fragment revealed that the deduced amino acid sequences contain 50 (B870 beta), 52 (B850 alpha), 283 (RCL) and 331 (RCM) residues with the corresponding molecular weights of 5592, 5814, 31364, and 37671, respectively. In the corresponding mRNA a 'hairpin' structure (delta G degrees = -26.6 kcal) is predicted to be located immediately downstream of pufA. The RC and LH polypeptides are highly homologous to those of the purple photosynthetic bacteria Rhodobacter capsulatus, Rhodobacter sphaeroides and Rhodopseudomonas viridis. Directly downstream of pufM there is an open reading frame (ORF) of unknown size. Partial sequencing indicates that this ORF is highly homologous to the cytochrome subunit of the photosynthetic reaction centre from R. viridis. In the puf operon no pufQ or pufX genes could be found, but the bchA gene is located upstream of that operon. Plasmid pESS8.9 containing the 2.728 kbp EcoRI fragment reconstituted a photoinactive mutant of Erythrobacter species OCH114. Comparative analysis of the DNA region upstream of the puf operon and of bacteriochlorophyll (Bchl) synthesis indicated that Bchl synthesis and puf gene expression are regulated differently in Erythrobacter and purple bacteria, respectively.     

Dark aerobic growth conditions induce the synthesis of a high midpoint potential cytochrome c8 in the photosynthetic bacterium Rubrivivax gelatinosus

In several strains of the photosynthetic bacterium Rubrivivax gelatinosus, the synthesis of a high midpoint potential cytochrome is enhanced 4-6-fold in dark aerobically grown cells compared with anaerobic photosynthetic growth. This observation explains the conflicting reports in the literature concerning the cytochrome c content for this species. This cytochrome was isolated and characterized in detail from Rubrivivax gelatinosus strain IL144. The redox midpoint potential of this cytochrome is +300 mV at pH 7. Its molecular mass, 9470 kDa, and its amino acid sequence, deduced from gene sequencing, support its placement in the cytochrome c8 family. The ratio of this cytochrome to reaction center lies between 0.8 and 1 for cells of Rvi. gelatinosus grown under dark aerobic conditions. Analysis of light-induced absorption changes shows that this high-potential cytochrome c8 can act in vivo as efficient electron donor to the photooxidized high-potential heme of the Rvi. gelatinosus reaction center.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

The influence of electrostatic interactions and intramolecular dynamics on electron transfer from the cytochrome subunit to the cation —radical of the bacteriochlorophyll dimer in reaction centers from Rps. viridis

Interheme electrostatic interaction can explain the acceleration of the electron transfer (ET) rate from the highest potential heme (C38o) to the photooxidized bacteriochlorophyll dimer (P⁺) which takes place after the reduction of neighbouring heme(s) of the cytochrome subunit in the reaction center of Rps. viridis. The electrostatic interaction energies calculated for neighbouring hemes, 7.0 Å apart (edge-to-edge), and for two high potential hemes, 21.5 Å apart are found to be 0.110 eV and 0.040 eV respectively. The reorganisation energy of the C₃₈₀-P⁺ transition of about 0.290±0.030 eV is calculated using the Marcus theory of electron tunneling. An empirical relation for the rate of ET is given. The low temperature restriction of the C₃₈₀-⁺ transition is caused by an energetic inhibition which originates from an opposite shifting of the energy levels of C₃₈₀ and P⁺ due to the freezing of protein dynamics and protein-bound water mobility. The freezing of the protein dynamics is revealed by the Mössbauer effect and correlates with the efficiency of the ET.Abbreviations RC reaction center - P⁺ cation-radical of bacteriochlorophyll dimer - C₃₈₀, C20, C310, C_–60, hemes indexed by the values of their individual redox potentials (in mV) - ET electron transfer     

Two distinct binding sites for high potential iron-sulfur protein and cytochrome c on the reaction center-bound cytochrome of Rubrivivax gelatinosus

The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways. A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark. A spontaneous reversion of R. gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth. This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette. We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis. We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome. Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8). Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.     

Interaction site for high-potential iron-sulfur protein on the tetraheme cytochrome subunit bound to the photosynthetic reaction center of Rubrivivax gelatinosus

We have recently demonstrated, using site-directed mutagenesis, that soluble cytochromes interact with the Rubrivivax gelatinosus photosynthetic reaction center (RC) in the vicinity of the low-potential heme 1 (c-551, Em = 70 mV) of the tetraheme cytochrome subunit, the fourth heme from the special pair of bacteriochlorophyll [Osyczka, A., et al. (1998) Biochemistry 37, 11732-11744]. Although the mutations generated in that study did not show clear effects on the electron transfer from high-potential iron-sulfur protein (HiPIP), which is the major physiological electron donor to the RC in this bacterium, we report here that other site-directed mutations near the solvent-exposed edge of the same low-potential heme 1, V67K (valine-67 substituted by lysine) and E79K/E85K/E93K (glutamates-79, -85, and -93, all replaced by lysines), considerably inhibit the electron transfer from HiPIP to the RC. Thus, it is concluded that HiPIP, like soluble cytochromes, binds to the RC in the vicinity of the exposed part of the low-potential heme 1 of the cytochrome subunit, although some differences in the configurations of the HiPIP-RC and cytochrome c-RC transient complexes may be postulated.