Complete amino acid sequence of cytochrome c551 from Erythrobacter species strain OCh 114

The complete amino acid sequence of cytochrome c551 isolated from an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114, was determined. The cytochrome molecule was composed of a total of 119 amino acid residues and its molecular weight including heme was calculated to be 13,235. The sequence was (Sequence: see text). Its molecular weight indicates that this cytochrome is of the L-type. Sequence alignment with other bacterial cytochromes c shows that this cytochrome is similar to cytochromes c of Rhodobacter capsulatus, Rhodobacter sphaeroides, and Paracoccus denitrificans, which were grouped into the alpha-3 subcluster from the 16S rRNA sequence analysis.     

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.     

Electron transfer proteins of the purple phototrophic bacterium, Rhodopseudomonas rutila.

The soluble electron transfer protein content of Rhodopseudomonas rutila was found to consist of two basic cytochromes and a (4Fe-4S) ferredoxin. Cytochrome c' was easily identified by its characteristic high spin absorption spectra. The native molecular weight is 29,000 and the subunit is 14,000. Cytochrome c-550 has low spin absorption spectra and a high redox potential (376 mV) typical of cytochromes c2. The molecular weight is about 14,000. The ferredoxin is apparently a dimer (43,000) of approximately 18,000 Da subunits. There are 1.3 to 1.5 iron-sulfur clusters per monomer of 18- to 21-kDa protein. The N-terminal amino acid sequence is like the (7Fe-8S) ferredoxins of Rhodobacter capsulatus and Azotobacter vinelandii. Remarkably, there are only 2 or 3 out of 25 amino acid substitutions. Difference absorption spectra of Rps. rutila membranes indicate that there is not tetraheme reaction center cytochrome c, such as is characteristic of Rps. viridis. However, there are a high potential cytochrome c and a low potential cytochrome b in the membrane, which are suggestive of a cytochrome bc1 complex. Rps. rutila is most similar to Rps. palustris in microbiological properties, yet it does not have the cytochromes c-556, c-554, and c-551 in addition to c2 and c', which are characteristic of Rps. palustris. Furthermore, the Rps. rutila cytochrome c' is dimeric, whereas the same protein from Rps. palustris is the only one known to be monomeric. The cytochrome pattern is more like that of Rhodospirillum rubrum and Rb. capsulatus, which are apparently only able to make cytochromes c2 and c'.     

Primary structure characterization of a Rhodocyclus tenuis diheme cytochrome c reveals the existence of two different classes of low-potential diheme cytochromes c in purple phototropic bacteria

The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cytochrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal insertions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting different functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious homology to the low-potential diheme cytochromes characterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cytochrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes.     

Cytochrome c2 mutants of Rhodobacter capsulatus.

Although structurally related to other members of the class I c-type cytochromes, the cytochromes c2 have little amino acid sequence homology to the eukaryotic cytochromes c. Moreover, the cytochromes c2 exhibit distinct properties such as redox potential and an isoelectric point. In an effort to understand the differences between the cytochromes c2 and the other class I c-type cytochromes, we have developed a genetic system to study Rhodobacter capsulatus cytochrome c2 by site-directed mutagenesis. We describe here overproduction of R. capsulatus wild-type cytochrome c2 in cytochrome c2-minus strains of R. capsulatus and Rhodobacter sphaeroides. We demonstrate that R. capsulatus wild-type cytochrome c2 can transcomplement for photosynthetic growth in R. sphaeroides. Further, we describe the generation, expression, and in vivo functionality properties of nine R. capsulatus site-directed mutants. We show that mutants K12D, K14E, K32E, K14E/K32E, P35A, W67Y, and Y75F are overproduced and functional in vivo. In contrast, mutants Y75C and Y75S are expressed at low levels and exhibit poor functionality in vivo. These findings establish an effective system for the production of R. capsulatus site-directed mutants and demonstrate that interspecies complementation can be used to detect defective cytochrome c2 mutants.     

A comparative study of the resonance Raman spectra of bacterial cytochromes

Resonance Raman spectra were obtained for two newly isolated bacterial cytochromes, Alcaligenes faecalis (ATCC 8750) c554 and Alcaligenes faecalis c556. Their spectra were compared with those of mammalian cytochrome c and two other bacterial cytochromes, Paracoccus denitrificans c550 and Pseudomonas aeruginosa c551. The positions of the Raman bands indicated that, while Al. c554 and Al. c556 were c-type cytochromes with two thioether linkages, several common features found in their Raman spectra were anomalous. These features suggest that the electronic charge density of the porphyrin macrocycle of Al. c554 and Al. c556 is more asymmetric than that of other bacterial and mammalian c-type cytochromes. The Raman evidence indicates that the electronic properties of the heme are controlled by the protein in these two Alcaligenes cytochromes.     

Protein dynamics: imidazole binding to class I C-type cytochromes.

The oxidized cytochrome c(2) from the purple phototrophic bacteria, Rhodobacter sphaeroides and Rhodobacter capsulatus, bind the neutral species of imidazole (K(a) = 1440 +/- 40 M(-1)) 50 times more strongly than does horse mitochondrial cytochrome c (K(a) = 30 +/- 1 M(-1)). The kinetics of imidazole binding are consistent with a change in rate-limiting step at high ligand concentrations for all three proteins. This is attributed to a conformational change leading to breakage of the iron-methionine bond which precedes imidazole binding. The three-dimensional structure of the Rb. sphaeroides cytochrome c(2) imidazole complex (Axelrod et al., Acta Crystalogr. D50, 596-602) supports the view that the conformational changes are essentially localized to approximately seven residues on either side of the ligated methionine and there is a hydrogen bond between the Phe 102 carbonyl, an internal water, and the bound imidazole. Insertions and deletions in this region of cytochrome c(2), the presence of a proline near the methionine, and the smaller size of the dynamic region of horse cytochrome c suggest that the stabilizing hydrogen bond is not present in horse cytochrome c, hence, the dramatic difference in affinity for imidazole. The kinetics of ligand binding do not correlate with either the strength of the iron-methionine bond as measured by the pK of the 695-nm absorption band or the overall stability of the cytochromes studied. However, the very similar imidazole binding properties of the two cytochromes c(2) indicate that the Rb. sphaeroides cytochrome c(2)-imidazole complex structure is an excellent model for the corresponding Rb. capsulatus cytochrome c(2) complex. It is notable that the movement of the peptide chain in the vicinity of the ligated methionine has been preserved throughout evolution and suggests a role in the function of c-type cytochromes.     

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.     

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.     

Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes.

We report the DNA sequence and mutational analysis of a novel cluster of six Bradyrhizobium japonicum genes of which at least three (designated cycV, cycW, and cycX) are essential for the formation of all cellular c-type cytochromes. Mutants having insertions in these genes were completely devoid of any soluble (periplasmic) or membrane-bound c-type cytochromes; even the apo form of cytochrome c1 was not detectable, neither in the membrane nor in the soluble fraction. As a consequence, the mutants had pleiotropic phenotypes such as defects in nitrate respiration, H2 oxidation, electron transport to cytochrome alpha alpha 3, and microaerobic respiration during symbiosis. A fourth open reading frame (ORF132) encoded a protein that might also be concerned with cytochrome c formation, but perhaps only indirectly. The other two open reading frames did not appear to function in this process. The predicted amino acid sequences of the cycW and cycX gene products suggested that these proteins were membrane-bound. The cycV gene product showed extensive similarity to the ATP-binding subunit of a superfamily of membrane-associated transport systems. The predicted ORF132 product was strikingly similar to bacterial thioredoxins and eukaryotic protein disulfide isomerase. Based on these findings it is possible that these proteins are members of a complex transport system involved in the biogenesis of all cytochromes c.     

Evidence from the structure and function of cytochromes c2 that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration

Cytochromes c₂ are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c₂ can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc₁ and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc₁ and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.     

Sponge-associated bacteria are strictly maintained in two closely related but geographically distant sponge hosts

The giant barrel sponges Xestospongia muta and Xestospongia testudinaria are ubiquitous in tropical reefs of the Atlantic and Pacific Oceans, respectively. They are key species in their respective environments and are hosts to diverse assemblages of bacteria. These two closely related sponges from different oceans provide a unique opportunity to examine the evolution of sponge-associated bacterial communities. Mitochondrial cytochrome oxidase subunit I gene sequences from X. muta and X. testudinaria showed little divergence between the two species. A detailed analysis of the bacterial communities associated with these sponges, comprising over 900 full-length 16S rRNA gene sequences, revealed remarkable similarity in the bacterial communities of the two species. Both sponge-associated communities include sequences found only in the two Xestospongia species, as well as sequences found also in other sponge species and are dominated by three bacterial groups, Chloroflexi, Acidobacteria, and Actinobacteria. While these groups consistently dominate the bacterial communities revealed by 16S rRNA gene-based analysis of sponge-associated bacteria, the depth of sequencing undertaken in this study revealed clades of bacteria specifically associated with each of the two Xestospongia species, and also with the genus Xestospongia, that have not been found associated with other sponge species or other ecosystems. This study, comparing the bacterial communities associated with closely related but geographically distant sponge hosts, gives new insight into the intimate relationships between marine sponges and some of their bacterial symbionts.     

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.     

Diversity of radA Genes from Cultured and Uncultured Archaea: Comparative Analysis of Putative RadA Proteins and Their Use as a Phylogenetic Marker

Archaea-specific radA primers were used with PCR to amplify fragments of radA genes from 11 cultivated archaeal species and one marine sponge tissue sample that contained essentially an archaeal monoculture. The amino acid sequences encoded by the PCR fragments, three RadA protein sequences previously published (21), and two new complete RadA sequences were aligned with representative bacterial RecA proteins and eucaryal Rad51 and Dmc1 proteins. The alignment supported the existence of four insertions and one deletion in the archaeal and eucaryal sequences relative to the bacterial sequences. The sizes of three of the insertions were found to have taxonomic and phylogenetic significance. Comparative analysis of the RadA sequences, omitting amino acids in the insertions and deletions, shows a cladal distribution of species which mimics to a large extent that obtained by a similar analysis of archaeal 16S rRNA sequences. The PCR technique also was used to amplify fragments of 15 radA genes from uncultured natural sources. Phylogenetic analysis of the amino acid sequences encoded by these fragments reveals several clades with affinity, sometimes only distant, to the putative RadA proteins of several species of Crenarcheota. The two most deeply branching archaeal radA genes found had some amino acid deletion and insertion patterns characteristic of bacterial recA genes. Possible explanations are discussed. Finally, signature codons are presented to distinguish among RecA protein family members.     

Molecular identification of Korean catfish (Siluriformes) based on two genetic markers

Eleven species of catfish (Siluriformes) found in Korean freshwater are economically important resources and include five endemic species. However, there are no studies on phylogenetic analysis of all catfish species in Korea at a molecular level. The species-level analysis of catfish species is usually carried out through morphological characters and controversial due to phenotypic variation. In this study, the partial sequences of 16S rRNA and cytochrome b mitochondrial genes were analyzed for species identification and phylogenetic relationships among 11 species of catfish from 10 different rivers in Korea. The nucleotide sequences of 16S rRNA and cytochrome b consisted of 587 and 441 nucleotide base pairs, respectively. Sequence analysis of both genes revealed that the 11 species fell into three distinct groups, which were genetically distinct from each other and exhibited identical phylogenetic resolution. Sequence divergences between congeneric species averaged 1.78% and 7.39% for 16S rRNA and cytochrome b, respectively. The phylogenetic relationships forming well-differentiated clades in the NJ, ML and BI trees were identical for both fragments. This research demonstrates that partial sequences of both the genes can efficiently identify the 11 species of catfish in Korea, indicating the usefulness of mtDNA-based approach in species identification.     

The primary structure of cytochrome c from the rust fungus Ustilago sphaerogena

1. The complete amino acid sequence of cytochrome c from the basidiomycete Ustilago sphaerogena was determined from the amino acid compositions and sequences of either tryptic or chymotryptic peptides, and in homology with at least thirty other established sequences of cytochrome c. 2. The primary structure of the molecule bears all of the characteristics of a mammalian-type cytochrome c, showing the typical clustered distribution of hydrophobic and basic residues with a single polypeptide chain of 107 residues. 3. Like all other fungal cytochromes c, it possesses a free N-terminus, and one less residue at the C-terminus than vertebrate cytochromes c. The region of residues 70-80 is strictly conserved, as is histidine at position 18. Position 26 is occupied by an asparagine residue, in contrast to histidine which occurs at this location in most of the known sequences of mammalian-type cytochromes c. 4. In contrast to some other fungal and plant cytochromes c of known primary structures, the Ustilago cytochrome c molecule does not contain trimethyl-lysine. 5. The sequence of Ustilago cytochrome c differs from the sequences of human, horse, chicken, tuna, wheat, and baker's yeast proteins at loci 47, 43, 44, 44 and 38 respectively.     

Controlling the functionality of cytochrome c(1) redox potentials in the Rhodobacter capsulatus bc(1) complex through disulfide anchoring of a loop and a beta-branched amino acid near the heme-ligating methionine.

The cytochrome c(1) subunit of the ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) contains a single heme group covalently attached to the polypeptide via thioether bonds of two conserved cysteine residues. In the photosynthetic bacterium Rhodobacter (Rba.) capsulatus, cytochrome c(1) contains two additional cysteines, C144 and C167. Site-directed mutagenesis reveals a disulfide bond (rare in monoheme c-type cytochromes) anchoring C144 to C167, which is in the middle of an 18 amino acid loop that is present in some bacterial cytochromes c(1) but absent in higher organisms. Both single and double Cys to Ala substitutions drastically lower the +320 mV redox potential of the native form to below 0 mV, yielding nonfunctional cytochrome bc(1). In sharp contrast to the native protein, mutant cytochrome c(1) binds carbon monoxide (CO) in the reduced form, indicating an opening of the heme environment that is correlated with the drop in potential. In revertants, loss of the disulfide bond is remediated uniquely by insertion of a beta-branched amino acid two residues away from the heme-ligating methionine 183, identifying the pattern betaXM, naturally common in many other high-potential cytochromes c. Despite the unrepaired disulfide bond, the betaXM revertants are no longer vulnerable to CO binding and restore function by raising the redox potential to +227 mV, which is remarkably close to the value of the betaXM containing but loop-free mitochondrial cytochrome c(1). The disulfide anchored loop and betaXM motifs appear to be two independent but nonadditive strategies to control the integrity of the heme-binding pocket and raise cytochrome c midpoint potentials.     

A multi‐locus approach resolves the phylogenetic relationships of the Simulium asakoae and Simulium ceylonicum species groups in Malaysia: evidence for distinct evolutionary lineages

A multi‐locus approach was used to examine the DNA sequences of 10 nominal species of blackfly in the Simulium subgenus Gomphostilbia (Diptera: Simuliidae) in Malaysia. Molecular data were acquired from partial DNA sequences of the mitochondria‐encoded cytochrome c oxidase subunit I (COI), 12S rRNA and 16S rRNA genes, and the nuclear‐encoded 18S rRNA and 28S rRNA genes. No single gene, nor the concatenated gene set, resolved all species or all relationships. However, all morphologically established species were supported by at least one gene. The multi‐locus sequence analysis revealed two distinct evolutionary lineages, conforming to the morphotaxonomically recognized Simulium asakoae and Simulium ceylonicum species groups.     

Complete amino acid sequence of cytochrome c from the honeybee, Apis mellifera, and evolutionary relationship of the honeybee to other insects on the basis of the amino acid sequence

The complete amino acid sequence of cytochrome c purified from the honeybee, Apis mellifera was determined. Only one molecular species of cytochrome c was found in the honeybee throughout its metamorphic stages. On the basis of a comparison of the amino acid sequence of honeybee cytochrome c with those of cytochromes c from other insects, it seems that the bee has evolutionarily appeared earlier than would be expected from the morphological and fossil evidence. If the classical phylogenetic relationships of the honeybee are correct, the evolutionary rate of cytochrome c must have been more rapid in the honeybee than in other insects.