Expression and characterization of the diheme cytochrome c subunit of the cytochrome bc complex in Heliobacterium modesticaldum

Heliobacterium modesticaldum is a Gram-positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b(6)f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc(1) complexes. One of the unique factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b(6)f complex or the monoheme cytochrome c(1) in the bc(1) complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c(4) proteins. The recombinant protein exhibits a single redox midpoint potential of +71mV versus NHE, which indicates that the two hemes have very similar protein environments.     

Expression, mutagenesis, and characterization of recombinant low-potential cytochrome c550 of photosystem II

Cytochrome c(550) of the photosystem II complex of cyanobacteria is an unusual member of the large protein family of monoheme c-type cytochromes. Despite the fact that it shares considerable amino acid sequence similarity and has a protein fold similar to the other members of the family, Cyt.c(550) has a midpoint potential (E(m7) = -250 mV) that is much lower than the positive midpoint potentials characteristic (E(m7) = 100-300mV) of this cytochrome family. An E. coli heterologous expression system involving secretion of the recombinant protein from Synechocystis PCC6803 to the periplasm was utilized to allow production of wild-type and mutant forms of the cytochrome. For most of the variants studied, the yield of protein was significantly enhanced by growth at 28 degrees C and inclusion of sucrose and betaine, in addition to isopropyl-beta-d-thiogalactoside (IPTG), to the growth medium of the E. coli expression host. Analysis of the protein products revealed that the wild-type protein maintained the redox and visible spectroscopic characteristics of the authentic protein. Mutations in the residues engaging in hydrogen bond interactions with the heme propionate (Asn49) and the axial 6th ligand His92 (Pro93) resulted in small (12-20 mV), but reproducible, upshifts in midpoint redox potential. Substitution of the axial ligand His92 with Met produced no discernible changes in the optical spectrum relative to the wild-type despite the fact that in this mutant, unlike the others studied here, the thioether linkage either was not formed or was highly labile as evidenced by loss of the heme during SDS-PAGE. On the other hand, the midpoint potential of the C550-H92M mutant was upshifted by approximately 70 mV. This value is significantly less of a perturbation than that observed in a similar mutant that is natively expressed in Thermosynechoccocus but appears to have an intact thioether linkage between the heme and the polypeptide moiety.     

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.     

Rapid electron transfer to photosystem I and unusual spectral features of cytochrome c(6) in Synechococcus sp. PCC 7002 in vivo

Cytochrome c(6) donates electrons to photosystem I (PS I) in Synechococcus sp. PCC 7002. In this work, we provide evidence for rapid electron transfer (t(1/2) = 3 micros) from cytochrome c(6) to PS I in this cyanobacterium in vivo, indicating prefixation of the reduced donor protein to the photosystem. We have investigated the cytochrome c(6)-PS I interaction by laser flash-induced spectroscopy of intact and broken cells and by redox titrations of membrane and supernatant fractions. Redox studies revealed the expected membrane-bound cytochrome f, b(6), and b(559) species and two soluble cytochromes with alpha-band absorption peaks of 551 and 553 nm and midpoint potentials of -100 and 370 mV, respectively. The characteristics and the symmetrical alpha-band spectrum of the latter correspond to typical cyanobacterial cytochrome c(6) proteins. Rapid oxidation of cytochrome c(6) by PS I in vivo results in a unique, asymmetric oxidation spectrum, which differs significantly from the spectra obtained for cytochrome c(6) in solution. The basis for the unusual cytochrome c(6) spectrum and possible mechanisms of cytochrome c(6) fixation to PS I are discussed. The occurrence of rapid electron transfer to PS I in cyanobacteria suggests that this mechanism evolved before the endosymbiotic origin of chloroplasts. Its selective advantage may lie in protection against photo-oxidative damage as shown for Chlamydomonas.     

Cytochrome cM from synechocystis 6803. Detection in cells, expression in Escherichia coli, purification and physical characterization.

Based on DNA sequence data a novel c-type cytochrome, cytochrome cM, has been predicted to exist in the cyanobacterium Synechocystis 6803. The precursor protein consists of 105 amino acids with a characteristic heme-binding motif and a hydrophobic domain located at the N-terminal end that is proposed to act as either a signal peptide or a membrane anchor. For the first time we report the detection of cytochrome cM in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome cM has been overexpressed in Escherichia coli in two forms, one with a poly histidine tag to facilitate purification and one without such a tag. The overexpressed protein has been purified and shown to bind heme, exhibiting an absorption peak in the Soret band near 416 nm and a peak in the alpha band at 550 nm. The extinction coefficient of cytochrome cM is 23.2 +/- 0.5 mM-1.cm-1 for the reduced minus oxidized alpha band peak (550-535 nm). The isoelectric point of cytochrome cM is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.2 predicted from the amino acid sequence. The redox midpoint potential of cytochrome cM expressed in E. coli is 151 +/- 5 mV (pH 7.1), which is quite low compared to other c-type cytochromes in which a histidine and a methionine residue serve as the axial ligands to the heme. This work opens the way for determining the three-dimensional structure of cytochrome cM and investigating its function in cyanobacteria.     

A new membrane-bound cytochrome c works as an electron donor to the photosynthetic reaction center complex in the purple bacterium, Rhodovulum sulfidophilum

A new type of membrane-bound cytochrome c was found in a marine purple photosynthetic bacterium, Rhodovulum sulfidophilum. This cytochrome c was significantly accumulated in cells growing under anaerobic photosynthetic conditions and showed an apparent molecular mass of approximately 100 kDa when purified and analyzed by SDS-PAGE. The midpoint potential of this cytochrome c was 369 mV. Flash-induced kinetic measurements showed that this new cytochrome c can work as an electron donor to the photosynthetic reaction center. The gene coding for this cytochrome c was cloned and analyzed. The deduced molecular mass was nearly equal to 50 kDa. Its C-terminal heme-containing region showed the highest sequence identity to the water-soluble cytochrome c(2), although its predicted secondary structure resembles that of cytochrome c(y). Phylogenetic analyses suggested that this new cytochrome c has evolved from cytochrome c(2). We, thus, propose its designation as cytochrome c(2m). Mutants lacking this cytochrome or cytochrome c(2) showed the same growth rate as the wild type. However, a double mutant lacking both cytochrome c(2) and c(2m) showed no growth under photosynthetic conditions. It was concluded that either the membrane-bound cytochrome c(2m) or the water-soluble cytochrome c(2) work as a physiological electron carrier in the photosynthetic electron transfer pathway of Rvu. sulfidophilum.     

The amino acid sequence of cytochrome c553 from Microcystis aeruginosa

Cytochrome c553 is an electron donor to P700 in the photosynthetic electron transfer chain of cyanobacteria and eukaryotic algae. We have purified this cytochrome from the cyanobacterium Microcystis aeruginosa and determined its amino acid sequence. When the amino acid sequence of this protein is compared to sequences of cytochromes c553 from other organisms, one sees that the evolution of net charge is more pronounced than the evolution of overall structure, further documenting a pronounced shift in the isoelectric point of this protein during the evolution of cyanobacteria. Cyanobacteria and algae also contain cytochrome c550 (Mr 15,500) which is quite different from cytochrome c553 (Mr 10,500). When the amino acid sequence of cytochrome c553 is compared to that of cytochrome c550, two regions of similar sequence are recognized.     

Structures of cytochrome c-549 and cytochrome c6 from the cyanobacterium Arthrospira maxima

Cytochrome c(6) and cytochrome c-549 are small (89 and 130 amino acids, respectively) monoheme cytochromes that function in photosynthesis. They appear to have descended relatively recently from the same ancestral gene but have diverged to carry out very different functional roles, underscored by the large difference between their midpoint potentials of nearly 600 mV. We have determined the X-ray crystal structures of both proteins isolated from the cyanobacterium Arthrospira maxima. The two structures are remarkably similar, superimposing on backbone atoms with an rmsd of 0.7 A. Comparison of the two structures suggests that differences in solvent exposure of the heme and the electrostatic environment of the heme propionates, as well as in heme iron ligation, are the main determinants of midpoint potential in the two proteins. In addition, the crystal packing of both A. maxima cytochrome c-549 and cytochrome c(6) suggests that the proteins oligomerize. Finally, the cytochrome c-549 dimer we observe can be readily fit into the recently described model of cyanobacterial photosystem II.     

Studies on algal cytochromes VI: some properties and amino acid sequence of cytochrome c6 from a green alga, Bryopsis maxima

A photosynthetic c-type cytochrome, cytochrome c6, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrcytochrome c6 has absorption maxima at 553.5 (alpha), 523 (beta), 417 (gamma), 318 (delta), and 275 nm, and the ferricytochrome at 695, 528, and 411 (gamma). The molecular weight was estimated to be about 10,000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferro- and ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferro- and ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9,904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKEDVTKYLDGGLSIEAITSQVRNGKGAMPAWSDRLD DEEIDGVVAYVFKNINEGW. A phylogenetic tree of 13 algal cytochromes c6 was constructed by comparing the amino acid differences.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.     

The Rieske/cytochrome b complex of Heliobacteria

Heliobacteria have a Rieske/cytochrome b complex composed of a Rieske protein, a cytochrome b(6,) a subunit IV and a di-heme cytochrome c. The overall structure of the complex seems close to the b(6)f complex from cyanobacteria and chloroplasts to the exception of the di-heme cytochrome. We show here by biochemical and biophysical studies that a heme c(i) is covalently attached to the Rieske/cytochrome b complex from Heliobacteria. We studied the EPR signature of this heme in two different species, Heliobacterium modesticaldum and Heliobacillus mobilis. In contrast to the case of b(6)f complex, a strong axial ligand to the heme is present, most probably a protonatable amino acid residue.     

In vivo photosystem I reduction in thermophilic and mesophilic cyanobacteria: the thermal resistance of the process is limited by factors other than the unfolding of the partners

Photosystem I reduction by plastocyanin and cytochrome c(6) in cyanobacteria has been extensively studied in vitro, but much less information is provided on this process inside the cell. Here, we report an analysis of the electron transfer from both plastocyanin and cytochrome c(6) to photosystem I in intact cells of several cyanobacterial species, including a comparative study of the temperature effect in mesophilic and thermophilic organisms. Our data show that cytochrome c(6) reduces photosystem I by following a reaction mechanism involving complex formation, whereas the copper-protein follows a simpler collisional mechanism. These results contrast with previous kinetic studies in vitro. The effect of temperature on photosystem I reduction leads us to conclude that the thermal resistance of this process is determined by factors other than the proper stability of the protein partners.     

Photoinduced electron transfer and cytochrome content in obligate aerobic phototrophic bacteria from genera Erythromicrobium,Sandaracinobacter, Erythromonas,Roseococcus and Erythrobacter

The photosynthetic apparatus and the electron carriers of seven species of five different genera of obligate aerobic phototrophic bacteria have been characterized by biochemical and biophysical techniques. A tetrahemic reaction center (RC) bound cytochrome (cyt) was found in Erythromonas (Em.) ursincola, Sandaracinobacter (S.) sibiricus and Roseococcus (R.) thiosulfatophilus, but not in Erythromicrobium (E.) ezovicum, Erythromicrobium ramosum, Erythromicrobium hydrolyticum and Erythrobacter (Eb.) litoralis. In none of the studied species, photochemical activity was observed under anaerobic conditions. Under aerobic conditions, the photoinduced cyclic electron transfer involves a soluble c-type cyt for the seven species. The cyt content of soluble and membrane fractions is highly dependent upon the species. The Erythromicrobium species (E. ezovicum, E. ramosum and E. hydrolyticum) contains a major soluble cyt while the other species possess several soluble cyts, up to four in the case of Eb. litoralis. These cyts have been characterized in terms of midpoint potential and apparent molecular mass. The presence of cyt bc₁ complexes has been clearly detected in Eb. litoralis, E. hydrolyticum, E. ezovicum and E. ramosum. These last three species also contain a high midpoint potential (350 mV) membrane-bound cyt c of unknown function.     

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c(6A)

Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

The cytochrome C oxidase genes in blue-green algae and characteristics of the deduced protein sequence for subunit II of the thermophilic cyanobacterium Synechococcus vulcanus.

Blue-green algae (cyanobacteria) contain both primitive photosynthetic and respiratory systems in their membranes. The controversial genes coding for an alpha alpha 3-type cytochrome oxidase in cyanobacteria were examined. The DNA probe coding for the most conserved part of subunit I hybridized with DNA fragments from four cyanobacterial species. We have cloned the genes coding for subunits I and II from the genomic library of the thermophilic cyanobacterium Synechococcus vulcanus and determined the nucleotide sequence of the subunit II gene. The deduced protein sequence (327 amino acid residues) indicates that there are two hydrophobic segments near the N-terminus and a hydrophilic intermembrane domain containing ligands for CuA (the ESR-active Copper) similar to other subunit IIs. The S. vulcanus subunit II does not contain the cytochrome c moiety that is present in bacilli and thermophiles.     

Purification and primary structure of cytochrome c-552 from the cyanobacterium, Synechococcus PCC 6312.

Cytochrome c-552 (soluble 'cytochrome f') from the unicellular cyanobacterium Synechococcus PCC 6312 (ATCC 27167) was purified and the primary structure determined. The proposed sequence consists of one polypeptide chain of 87 residues. The sequence was determined by a combination of chemical and enzymatic cleavage, manual and automatic sequencing and mass spectroscopy. This is the first amino acid sequence of this cytochrome from a unicellular cyanobacterium to be determined in a study of the variation in primary structure between phylogenetically distant cyanobacteria. The sequence is compared to the primary structures of the cytochrome from filamentous cyanobacteria and from eukaryotic algae. The significance of these sequence comparisons to the current hypotheses concerning the origin of eukaryotic cells and their chloroplasts is discussed.     

Structure of the soluble domain of cytochrome f from the cyanobacterium Phormidium laminosum.

Cytochrome f from the photosynthetic cytochrome b(6)f complex is unique among c-type cytochromes in its fold and heme ligation. The 1. 9-A crystal structure of the functional, extrinsic portion of cytochrome f from the thermophilic cyanobacterium Phormidium laminosum demonstrates that an unusual buried chain of five water molecules is remarkably conserved throughout the biological range of cytochrome f from cyanobacteria to plants [Martinez et al. (1994) Structure 2, 95-105]. Structure and sequence conservation of the cytochrome f extrinsic portion is concentrated at the heme, in the buried water chain, and in the vicinity of the transmembrane helix anchor. The electrostatic surface potential is variable, so that the surface of P. laminosum cytochrome f is much more acidic than that from turnip. Cytochrome f is unrelated to cytochrome c(1), its functional analogue in the mitochondrial respiratory cytochrome bc(1) complex, although other components of the b(6)f and bc(1) complexes are homologous. Identical function of the two complexes is inferred for events taking place at sites of strong sequence conservation. Conserved sites throughout the entire cytochrome b(6)f/bc(1) family include the cluster-binding domain of the Rieske protein and the heme b and quinone-binding sites on the electrochemically positive side of the membrane within the b cytochrome, but not the putative quinone-binding site on the electrochemically negative side.     

Cytochrome C553 from Desulfovibrio,vulgaris: Potentiometric characterization by optical and EPR studies

Cytochrome c₅₅₃ is a monohaemic c type cytochrome isolated from the sulfate reducing bacteria $\text{Desulfovibrio}$,$\text{vulgaris}$. Its midpoint potential value, determined by optical, EPR and polarographic studies is significantly lower than the midpoint potentials reported for other monohaemic cytochromes c (+ 10 mV instead of + 290 mV). In an attempt to study correlations between amino acid sequence, haem iron coordination and haem exposure in cytochromes c, cytochrome c₅₅₃ is compared with mitochondrial and bacterial c type cytochromes.