The structure of Paracoccus denitrificans cytochrome c550.

The crystal structure of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been solved by x-ray diffraction to a resolution of 2.45 A. In both amino acid sequence and molecular structure it is evolutionarily homologous with mitochondrial cytochrome c from eukaryotes and photosynthetic cytochrome c2 from purple non-sulfur bacteria. All of these cytochromes c have the same basic folding pattern, with surface insertions of extra amino acids in c550. Various strains of c2 have all, some, or none of the extra insertions observed in c550. The hydrophobic heme environment, position of aromatic rings, and structure and environment of the heme crevice, are virtually identical in cytochromes c55o, c, and c2. Radical changes observed at all regions on the molecular surface except the heme crevice argue for the importance of the crevice and the exposed edge of the heme in the transfer of electrons to and from the cytochrome molecule.     

Structural homology of cytochromes c.

Cytochromes c from many eukaryotic and diverse prokaryotic organisms have been investigated and compared using high-resolution nuclear magnetic resonance spectroscopy. Resonances have been assigned to a large number of specific groups, mostly in the immediate environment of the heme. This information, together with sequence data, has allowed a comparison of the heme environment and protein conformation for these cytochromes. All mitochondrial cytochromes c are found to be very similar to the cytochromes c2 from Rhodospirillaceae. In the smaller bacterial cytochromes, Pseudomonas aeruginosa cytochrome c551 and Euglena gracilis cytochrome c552, the orientation of groups near the heme is very similar, but the folding of the polypeptide chain is different. The heme environment of these two proteins is similar to that of the larger bacterial and mitochondrial cytochromes. Two low-potential cytochromes, Desulfovibrio vulgaris cytochrome c553 and cytochrome c554 from a halotolerant micrococcus have heme environments which are not very similar to those of the other proteins reported here.     

Structural and biochemical characterization of DHC2, a novel diheme cytochrome c from Geobacter sulfurreducens

Multiheme cytochromes c constitute a widespread class of proteins with essential functions in electron transfer and enzymatic catalysis. Their functional properties are in part determined by the relative arrangement of multiple heme cofactors, which in many cases have been found to pack in conserved interaction motifs. Understanding the significance of these motifs is crucial for the elucidation of the highly optimized properties of multiheme cytochromes c, but their spectroscopic investigation is often hindered by the large number and efficient coupling of the individual centers and the limited availability of recombinant protein material. We have identified a diheme cytochrome c, DHC2, from the metal-reducing soil bacterium Geobacter sulfurreducens and determined its crystal structure by the method of multiple-wavelength anomalous dispersion (MAD). The two heme groups of DHC2 pack into one of the typical heme interaction motifs observed in larger multiheme cytochromes, but because of the absence of further, interfering cofactors, the properties of this heme packing motif can be conveniently studied in detail. Spectroscopic properties (UV-vis and EPR) of the protein are typical for cytochromes containing low-spin Fe(III) centers with bis-histidinyl coordination. Midpoint potentials for the two heme groups have been determined to be -135 and -289 mV by potentiometric redox titrations. DHC2 has been produced by recombinant expression in Escherichia coli using the accessory plasmid pEC86 and is therefore accessible for systematic mutational studies in further investigating the properties of heme packing interactions in cytochromes c.     

Cytochrome c maturation. The in vitro reactions of horse heart apocytochrome c and Paracoccus dentrificans apocytochrome c550 with heme

C-type cytochromes are characterized by having the heme moiety covalently attached via thioether bonds between the heme vinyl groups and the thiols of conserved cysteine residues of the polypeptide chain. Previously, we have shown the in vitro formation of Hydrogenobacter thermophilus cytochrome c(552) (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). In this work we report that thioether bonds can form spontaneously in vitro between heme and the apocytochromes c from horse heart and Paracoccus denitrificans via b-type cytochrome intermediates. Both apocytochromes, but not the holo forms, bind 8-anilino-1-naphthalenesulfonate, indicating that the apoproteins each have an affinity for a hydrophobic ligand. Furthermore, for both apocytochromes c an intramolecular disulfide can form between the cysteines of the CXXCH motif that is characteristic of c-type cytochromes. In vitro reaction of these apocytochromes c with heme to yield holocytochromes c, and the tendency to form a disulfide, have implications for the different systems responsible for cytochrome c maturation in vivo in various organisms.     

Stereoselective in vitro formation of c-type cytochrome variants from Hydrogenobacter thermophilus containing only a single thioether bond

In vitro formation of Hydrogenobacter thermophilus cytochrome c552 has previously been demonstrated (Daltrop, O., Allen, J. W. A., Willis, A. C., and Ferguson, S. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7872-7876). Now we report that the single cysteine variants of H. thermophilus c552, which bind heme via a single thioether bond, also form in vitro. Furthermore, reaction of the apocytochromes containing either AXXCH or CXXAH in the binding motif with 2-vinyldeuteroheme and 4-vinyldeuteroheme resulted predominantly in covalent attachment between Cys-11 and the 2-vinyl moiety and Cys-14 and the 4-vinyl functionality. This observation shows that the covalent attachment of heme to apocytochrome is stereoselective, indicating that the initial non-covalent complexes between apoprotein and heme have to be in the correct stereochemical orientation for preferential promotion of thioether bond formation. Additionally, the heme derivatives 2-vinyldeuteroheme and 4-vinyldeuteroheme were reacted with wild-type H. thermophilus c552 to yield another modification of cytochromes containing only one thioether bond. These results show that the formation of the two thioether bonds in typical c-type cytochromes can occur independently from one another. Aspects of rotational isomerism of heme in heme-proteins are discussed.     

Cytochrome c biogenesis in mitochondria--Systems III and V

In c-type cytochromes, heme becomes covalently attached to the polypeptide chain by a reaction between the vinyl groups of the heme and cysteine thiols from the protein. There are two such cytochromes in mitochondria: cytochrome c and cytochrome c(1). The heme attachment is a post-translational modification that is catalysed by different biogenesis proteins in different organisms. Three types of biogenesis system are found or predicted in mitochondria: System I (the cytochrome c maturation system); System III (termed holocytochrome c synthase (HCCS) or heme lyase); and System V. This review focuses primarily on cytochrome c maturation in mitochondria containing HCCS (System III). It describes what is known about the enzymology and substrate specificity of HCCS; the role of HCCS in human disease; import of HCCS into mitochondria; import of apocytochromes c and c(1) into mitochondria and the close relationships with HCCS-dependent heme attachment; and the role of the fungal cytochrome c biogenesis accessory protein Cyc2. System V is also discussed; this is the postulated mitochondrial cytochrome c biogenesis system of trypanosomes and related organisms. No cytochrome c biogenesis proteins have been identified in the genomes of these organisms whose c-type cytochromes also have a unique mode of heme attachment.     

Site-directedly mutated human cytochrome c which retains heme c via only one thioether bond

Although Cys-14 (human numbering) of cytochrome c was conserved during its molecular evolution and it is supposed to be essential for most cytochromes c to retain heme c via two thioether bonds, a site-directedly mutated human cytochrome c which has an alanine residue at this position and only one thioether bond through Cys-17 turns out to be functional. This shows that Cys-14 is not essential. The absorption spectrum of the atypical cytochrome c is red shifted, and similar to those of Euglena and Crithidia cytochromes c, which also have only one thioether bond [Pettigrew, G.W., Leaver, J.L., Meyer, T.E., & Ryle, A.P. (1975) Biochem. J. 147, 291-302].     

Order within a mosaic distribution of mitochondrial c-type cytochrome biogenesis systems?

Mitochondrial cytochromes c and c(1) are present in all eukaryotes that use oxygen as the terminal electron acceptor in the respiratory chain. Maturation of c-type cytochromes requires covalent attachment of the heme cofactor to the protein, and there are at least five distinct biogenesis systems that catalyze this post-translational modification in different organisms and organelles. In this study, we use biochemical data, comparative genomic and structural bioinformatics investigations to provide a holistic view of mitochondrial c-type cytochrome biogenesis and its evolution. There are three pathways for mitochondrial c-type cytochrome maturation, only one of which is present in prokaryotes. We analyze the evolutionary distribution of these biogenesis systems, which include the Ccm system (System I) and the enzyme heme lyase (System III). We conclude that heme lyase evolved once and, in many lineages, replaced the multicomponent Ccm system (present in the proto-mitochondrial endosymbiont), probably as a consequence of lateral gene transfer. We find no evidence of a System III precursor in prokaryotes, and argue that System III is incompatible with multi-heme cytochromes common to bacteria, but absent from eukaryotes. The evolution of the eukaryotic-specific protein heme lyase is strikingly unusual, given that this protein provides a function (thioether bond formation) that is also ubiquitous in prokaryotes. The absence of any known c-type cytochrome biogenesis system from the sequenced genomes of various trypanosome species indicates the presence of a third distinct mitochondrial pathway. Interestingly, this system attaches heme to mitochondrial cytochromes c that contain only one cysteine residue, rather than the usual two, within the heme-binding motif. The isolation of single-cysteine-containing mitochondrial cytochromes c from free-living kinetoplastids, Euglena and the marine flagellate Diplonema papillatum suggests that this unique form of heme attachment is restricted to, but conserved throughout, the protist phylum Euglenozoa.     

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.     

Proton and nitrogen-15 NMR studies of ferricytochrome c cyanide complexes: remarkable conservation of the heme environment among organisms of diverse origin

The native ferric and cyanide-bound ferric forms of nine vertebrate and two yeast cytochromes c have been investigated by high-resolution proton nuclear magnetic resonance spectroscopy. Spectral comparisons have been made among the cytochromes with emphasis on the signal positions for heme and amino acid ligand protons. Consistent with earlier more limited studies of native ferric cytochromes c, the paramagnetically shifted proton NMR signals show little variation among species with up to 50% substitution of amino acids. Proton NMR spectra for the cyanide complexes also show little variation among species. The nitrogen-15 signal for the coordinated cyanide ion is known to be highly variable among other hemoproteins, but the signal covers a range of only 855 to 865 ppm (nitrate ion reference) for vertebrate cytochromes c and 884 to 886 ppm for yeast cytochromes c. The cyanide ligand probe thus reports an amazing conservation of the heme and proximal ligand environment among the cytochromes. Comparative proton and nitrogen-15 chemical shift values are consistent with a slightly stronger proximal histidine imidazole hydrogen bond to an amino acid carbonyl function than is the case for hemoglobin and myoglobin.     

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.     

Resonance Raman study of multihemic c-type cytochromes from Desulfuromonas acetoxidans.

Two multihemic cytochromes c from the sulfur reducing bacteria Desulfuromonas acetoxidans have been studied by optical and resonance Raman spectroscopy: cytochrome c551.5, a trihemic cytochrome and cytochrome c Mr 50 000, a recently isolated high molecular mass cytochrome. The redox and Raman characteristics of cytochrome c551.5 are compared to those of the tetrahemic cytochromes c3 from Desulfovibrio. While the redox behavior, followed by spectroelectrochemistry, is similar to that of cytochrome c3, showing the same conformational change after reduction of the highest potential heme, the Raman data show a contribution from a His- form of the axial ligands and lead to the assignment of a band at 218 cm-1 to the Fe(III)-(His)2 stretching vibration. The Raman data on cytochrome c Mr 50 000 are in favor of an entirely low spin species with two different sets of axial ligands. A partially reduced state is easily accessible by ascorbate addition.     

The solution structure of a tetraheme cytochrome from Shewanella frigidimarina reveals a novel family structural motif

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 ( Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c 3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis ( Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.     

Design and synthesis of de novo cytochromes c

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.     

Structure of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus: comparative study on the thermostability of cytochrome c

We have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; Pseudomonas aeruginosa (PAc(551)) growing at 37 degrees C and Hydrogenobacter thermophilus (HTc(552)) at 72 degrees C and showed that only five residues primarily differentiate their stabilities. For a more comprehensive study, we found Hydrogenophilus thermoluteolus (Pseudomonas hydrogenothermophila) growing at 52 degrees C and showed the moderate stability of the cytochrome c from this bacterium (PHc(552)). To explore the stabilization mechanisms, the crystal structure of PHc(552) was determined by X-ray analysis. The solution structure of HTc(552) elucidated previously by NMR was refined using distributed computational implementation. Furthermore, the recently reported crystal structure of HTc(552) has become available [Travaglini-Allocatelli, C. et al. (2005) J. Biol. Chem. 280, 25729-25734]. When the structures of these three cytochromes c were combined, this revealed that the five residues, corresponding to those mentioned above, determine the difference of stabilities among them as well. These facts suggested the stabilization mechanisms as follows: (1) improved van der Waals interactions by packing optimization at the N-terminal helix, (2) attractive electrostatic interactions with the heme propionate group, and (3) favorable van der Waals interaction with the heme. This comparative study, by supplementing the structural information of PHc(552) with its complementary feature, demonstrates that just a small number of amino acid residues determine the overall molecular stability by means of additivity of the effects of their substitutions. It is interesting that, in naturally occurring proteins, these adaptation strategies are accommodated by these bacteria to survive in the wide range of thermal conditions.     

Control of DegP-dependent degradation of c-type cytochromes by heme and the cytochrome c maturation system in Escherichia coli

c-Type cytochromes are located partially or completely in the periplasm of gram-negative bacteria, and the heme prosthetic group is covalently bound to the protein. The cytochrome c maturation (Ccm) multiprotein system is required for transport of heme to the periplasm and its covalent linkage to the peptide. Other cytochromes and hemoglobins contain a noncovalently bound heme and do not require accessory proteins for assembly. Here we show that Bradyrhizobium japonicum cytochrome c550 polypeptide accumulation in Escherichia coli was heme dependent, with very low levels found in heme-deficient cells. However, apoproteins of the periplasmic E. coli cytochrome b562 or the cytosolic Vitreoscilla hemoglobin (Vhb) accumulated independently of the heme status. Mutation of the heme-binding cysteines of cytochrome c550 or the absence of Ccm also resulted in a low apoprotein level. These levels were restored in a degP mutant strain, showing that apocytochrome c550 is degraded by the periplasmic protease DegP. Introduction of the cytochrome c heme-binding motif CXXCH into cytochrome b562 (c-b562) resulted in a c-type cytochrome covalently bound to heme in a Ccm-dependent manner. This variant polypeptide was stable in heme-deficient cells but was degraded by DegP in the absence of Ccm. Furthermore, a Vhb variant containing a periplasmic signal peptide and a CXXCH motif did not form a c-type cytochrome, but accumulation was Ccm dependent nonetheless. The data show that the cytochrome c heme-binding motif is an instability element and that stabilization by Ccm does not require ligation of the heme moiety to the protein.     

Exploration of the 'cytochromome' of Desulfuromonas acetoxidans, a marine bacterium capable of powering microbial fuel cells

Recent progress in bacterial genomic analysis has revealed a vast number of genes that encode c-type cytochromes that contain multiple heme cofactors. This high number of multiheme cytochromes in several bacteria has been correlated with their great respiratory flexibility, and in what concerns biotechnological applications, has been correlated with electricity production in Microbial Fuel Cells. Desulfuromonas acetoxidans, a member of the Geobactereaceae family, is one of these organisms for which the genome was recently made available, coding for 47 putative multiheme cytochromes. The growth of D. acetoxidans in different media allowed the identification of the cytochromes dominant in each condition. The triheme cytochrome c(7) is always present suggesting a key role in the bioenergetic metabolism of this organism, and a dodecaheme cytochrome of low homology with other proteins in the databases was also isolated. Different cytochromes are found for different growth conditions showing that their roles can be assigned to specific bioenergetic electron transfer routes.     

Solution structure and characterization of the heme chaperone CcmE

The covalent attachment of the heme cofactor in c-type cytochromes is a surprisingly complex process, which in bacteria involves a number of different proteins. Among the latter, the ccmE gene product is known to perform a key role in the heme delivery pathway in Gram-negative bacteria. The solution structure of the soluble domain of apo-CcmE from Shewanella putrefaciens was determined through NMR spectroscopy on a 13C,15N-labeled sample. The structure is characterized by a compact core with large regions of beta structure, while the N-terminal and C-terminal regions are essentially unstructured. The overall folding is similar to that of the so-called oligo-binding proteins (OB fold). Solvent-exposed aromatic residues, conserved in all CcmE homologues, have been found in the proximity of His131, the putative heme-binding residue, that could have a role in the interaction with heme. No interaction between CcmE and heme, as well as between CcmE and holocytochrome c, could be detected in vitro by electronic spectroscopy or by NMR. The data available suggest that the heme transfer process is likely to involve a heterooligomeric protein complex and occur under a tight enzymatic control.     

Bacillus subtilis 13-kilodalton cytochrome c-550 encoded by cccA consists of a membrane-anchor and a heme domain

Little is known about c-type cytochromes in Gram-positive bacteria in contrast to the wealth of information available on this type of cytochrome in Gram-negative bacteria and in eucaryotes. In the present work, the strictly aerobic bacterium Bacillus subtilis was analyzed for subcellular localization and number of different cytochromes c. In vivo labeling with radioactive 5-aminolevulinic acid, a precursor to heme, showed that the proteins containing covalently bound heme are predominantly found in the membrane fraction. One major membrane-bound cytochrome c of about 15 kDa and with an alpha-band absorption peak in the reduced state at 550 nm was analyzed in more detail. Cytochrome c-550 has the properties of an integral membrane protein. The physiological function of this relatively high redox potential cytochrome is not known. Its structural gene, cccA, was cloned, sequenced, and overexpressed in B. subtilis. The gene maps adjacent to rpoD (sigA) at 223 degrees on the chromosome. The amino acid sequence of cytochrome c-550 as deduced from the DNA sequence consists of 120 residues and contains one heme c binding site (Cys-Ile-Ala-Cys-His) located approximately in the middle of the polypeptide. From the hydropathy distribution and from comparisons to soluble c-type cytochromes of known three-dimensional structure, cytochrome c-550 seemingly consists of two domains; an N-terminal membrane-anchor domain and a C-terminal heme domain. A model for the topography of the cytochrome in the cytoplasmic membrane is suggested in which the N-terminal part spans the membrane in the form of a single segment in an alpha-helical conformation and the C-terminal heme domain is exposed on the extracytoplasmic side of the membrane. Deletion of cccA from the chromosome revealed another membrane-bound cytochrome with absorption maximum at 550 nm in the reduced state. Analysis of cccA deletion mutants demonstrated that the cytochrome c-550 encoded by cccA is not essential for growth of B. subtilis on rich or minimal media.     

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.