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.     

The amino acid sequence of cytochrome c of Fagopyrum esculentum Moench (buckwheat) and Brassica oleracea L. (cauliflower)

The amino acid sequences of buckwheat and cauliflower cytochromes c were determined on 1(1/2)mumol and 1mumol of protein respectively. The molecules consist of 111 residues and are homologous with other plant mitochondrial cytochromes c. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7.     

The dipole moment of cytochrome c.

Vertebrate cytochromes c and the cytochromes c of insects and plants have, on average, dipole moments of 320 and 340 debye, respectively. The direction of the dipole vector with respect to the haem plane, at the solvent-accessible edge of which electron transfer presumably takes place, is conserved in these two groups--at 32 degrees +/- 7 degrees and 22 degrees +/- 10 degrees, respectively. The variation of dipole orientations and magnitudes observed in these species is compared with the results of a model in which charge distributions occur randomly. Since this model does not generate the observed charge asymmetries of the various cytochromes c, it is concluded that the dipole moment of cytochrome c is a feature that is evolutionarily conserved, apparently because it has an important influence on the interaction of this mobile electron carrier with its physiological electron donors and acceptors in the intermembrane space of mitochondria.     

Characterization of cytochromes from Methanosarcina strain Göl and their involvement in electron transport during growth on methanol.

Methanosarcina strain G?1 was tested for the presence of cytochromes. Low-temperature spectroscopy, hemochrome derivative spectroscopy, and redox titration revealed the presence of two b-type (b559 and b564) and two c-type (c547 and c552) cytochromes in membranes from Methanosarcina strain G?1. The midpoint potentials determined were Em,7 = -135 +/- 5 and -240 +/- 11 mV (b-type cytochromes) and Em,7 = -140 +/- 10 and -230 +/- 10 mV (c-type cytochromes). The protoheme IX and the heme c contents were 0.21 to 0.24 and 0.09 to 0.28 mumol/g of membrane protein, respectively. No cytochromes were detectable in the cytoplasmic fraction. Of various electron donors and acceptors tested, only the reduced form of coenzyme F420 (coenzyme F420H2) and the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate (CoM-S-S-HTP) were capable of reducing and oxidizing the cytochromes at a high rate, respectively. Addition of CoM-S-S-HTP to reduced cytochromes and subsequent low-temperature spectroscopy revealed the oxidation of cytochrome b564. On the basis of these results, we suggest that one or several cytochromes participate in the coenzyme F420H2-dependent reduction of the heterodisulfide.     

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.     

Kinetic and equilibrium studies of alkaline isomerization of vertebrate cytochromes c

Equilibria and kinetics of alkaline isomerization of seven ferricytochromes c from vertebrates were studied by pH-titration and pH-jump methods in the pH region of 7-12. In the equilibrium behavior, no significant difference was detected among the cytochromes c, whereas marked differences in the kinetic behavior were observed. According to the kinetic behavior of the isomerization, the cytochromes c examined fall into three classes: Group I (horse, sheep, dog and pigeon cytochromes c), Group II (tuna and bonito cytochromes c) and Group III (rhesus monkey cytochrome c). The kinetic results are interpreted in terms of the sequential scheme: Neutral form in equilibrium with fast Transient form in equilibrium with slow Alkaline form where the neutral and alkaline forms are the species stable at neutral and alkaline pH, respectively, and the transient form is a kinetic intermediate. From comparison of the primary sequences of the seven cytochromes c and the classification of these cytochromes c, it is concluded that the amino acid substitution Phe/Tyr at the 46-th position has a major influence on the kinetic behavior. In Group II and III cytochromes c, the ionization of Tyr-46 is suggested to bring about loosening of the heme crevice and thus facilitate the ligand replacement involved in the isomerization.     

The amino acid sequences of the cytochromes c-555 from two green sulphur bacteria of the genus Chlorobium.

Amino acid seauences are proposed for the cytochromes c-555 from Chlorobium thiosulphatophilum and from the Chlorobium limicola component of "Chloropseudomonas ethylica 2K". Each is a sincle polypeptide chain, the former of 86, the latter of 99 residues, and, when aligned so as to give the best match, 47 residues are common to the two sequences. The sequences show some resemblance to those of cytochromes c5 and f. The bacteriochlorophyll a-proteins were also isolated and purified, and their amino acid compositions compared (see the Appendix). There are significant differences in the compositions, but not as great as those found for the cytochromes c-555. The significance of these observations for the taxonomy of the Chlorobiaceae and for the further development of the comparative biochemistry of cytochrome c is discussed. Detailed evidence for the sequences of the cytochromes c-555 has been deposited as Supplementary Publication SUP 50073 (36 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1976) 153, 5.     

The amino acid sequence of cytochrome c from Nigella damascena L. (love-in-a-mist)

The amino acid sequence of cytochrome c from Nigella damascena L. was determined on 0.2mumol of protein. Peptides from a single chymotryptic digest were analysed by the dansyl-Edman procedure. These peptides were ordered by reference to the sequences of other plant cytochromes c, assuming that the Nigella cytochrome is homologous with the other cytochromes. Many of the Nigella peptides were one or two residues short when compared with the corresponding chymotryptic peptides from other plant cytochromes c. These residues are assumed to have been removed by an endogenous carboxypeptidase, and the identification and placing of these residues is entirely based on homology. These residues are numbered 3, 18, 42, 43, 44, 54, 67, 72, 73, 82 and 105. A number of other positions are almost entirely placed by homology. These are positions which could not be placed definitely by dansyl-Edman analysis or by dansylation after digestion with carboxypeptidase A, and are numbered 14, 15, 16, 39, 40, 85, 86, 87 and 88. Except for residue 15, all residues based entirely, or nearly so, on homology have been previously found invariant in sequences of plant cytochromes c. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50017, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

Equilibrium and kinetic studies of unfolding of homologous cytochromes c.

Extensive investigations of the unfolding equilibria and kinetics of oxidized and reduced cytochromes c are reported. It is found that all cytochromes c have similar unfolding free energies (deltaGD = 7 +/- 1 kcal/mol). Differences among species do not correlate in any way with the metabolic differences among species. The stabilization of cytochrome c on reduction is estimated at 1.1 kcal/mol. Stability differences among species are mirrored in their denaturation kinetics. For cytochrome c (III), the unfolding exhibits multiple phases. The rate constants for the two observable phases both change by a factor of 3 between horse cytochrome c (III) and cow cytochrome c (III). On reduction, all unfolding appears to occur in a single step. The rate of this unfolding still varies between species, however, the results can be accommodated to a sequential model, with some assumptions. The observations are consistent with chain reversal occurring at an early stage in the reaction and suggest that previously observed rapid processes may be ligand exchange processes.     

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'.     

Deeply branching c6-like cytochromes of cyanobacteria

The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.     

bc1-Complex from beef heart. One-step purification by hydroxyapatite chromatography in Triton X-100, polypeptide pattern and respiratory chain characteristics.

A new simple method for the purification of the bc1-complex has been developed. The polypeptide composition of the complex was analysed by dodecyl sulfate-polyacrylamide gel electrophoresis. The content of chain components and phospholipids was determined. The b-type cytochromes were further characterized by their absorbance spectra and midpoint potentials. (1) Starting from a Triton X-100 extract of submitochondrial particles supplemented with antimycin, the bc1-complex is purified by adsorption chromatography on hydroxyapatite with citrate as specific eluant. (2) The complex splits in dodecyl sulfate into five main polypeptides with apparent molecular weight of 47, 44, 31, 11 and less than 10 kdalton. (3) The purified complex has a heme-b content of 8.0 mumol/g protein and a cytochrome c1 content of 3.8 mumol/g protein. (4) The cytochromes show the typical absorbance spectra of cytochromes b-562 and b-565 and are present in approximately equal amounts with midpoint potentials of Em7 = + 100 mV and Em7 = + mV respectively. Carbon monoxide does not bind to the cytochromes. (5) The nonheme iron protein content of the complex is diminished to 0.6 mumol/g protein. (6) The use of the nonionic surfactant Triton X-100 leads to a complete loss of lipids and ubiquinone of the bc1-complex. (7) The complex contains no succinate dehydrogenase as indicated by the absence of the 69 kdalton subunit in the dodecyl sulfate gel electrophoresis. In addition, it lacks an ubiquinone cytochrome c reductase activity and other electron transferring activities. This may be inferred from an inhibition by antimycin and depletion of ubiquinone and phospholipids. The highly purified and relative stable complex can be prepared giving 50% yield and may be suitable for protein chemistry studies.     

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.     

The amino acid sequence of cytochrome c from Cucurbita maxima L. (pumpkin)

The amino acid sequence of pumpkin cytochrome c was determined on 2mumol of protein. Some evidence was found for the occurrence of two forms of cytochrome c, whose sequences differed in three positions. Pumpkin cytochrome c consists of 111 residues and is homologous with mitochondrial cytochromes c from other plants. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50005 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1971), 121, 7.     

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.     

The amino acid sequence of cytochrome c from the locust, Schistocerca gregaria Forskal.

The amino acid sequence of locust cytochrome c was determined, although the overlap between chymotryptic and tryptic peptides at residues tyrosine-97 and leucine-98 was not observed, owing to an anomalous tryptic break duplicating the chymotryptic digestion. The molecule consists of a single polypeptide chain of 107 residues, homologous with other mitochondrial cytochromes c. In common with other known insect cytochromes c, it possesses a non-acetylated, four-residue tail at the N-terminus relative to glycine-1 of the standard alignment. A molecular phylogeny for 17 species was constructed relating the cytochrome c molecules of Schistocerca gregaria and other invertebrates with those of representative taxonomic groups. Experimental details are given in a supplementary paper deposited as Supplementary Publication SUP 50077 (24 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can obtained on the terms indicated in Biochem. J. (1977) 161, 1.     

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.     

Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12 nm protein with interacting hemes

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2? resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15? resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c(7)-type domains (A, B, C and D) with homology to triheme cytochromes c(7). In cytochromes c(7) all three hemes are bis-His coordinated, whereas in c(7)-type domains the last heme is His-Met coordinated. The full-length GSU1996 has a 12nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a "nanowire" of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c(7)-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c(7)-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c(7)-type domains from one multiheme protein were compared.     

Structure of bonito heart ferricytochrome c and some remarks on molecular interaction in its crystalline state.

The structure analysis of bonito heart ferricytochrome c was carried out at 2.8 A resolution by X-ray diffraction. The overall features of the molecule are virtually identical with those of bonito ferrocytochromes c and other cytochromes c. In the present work, the modes of molecular packing among cytochromes c were also compared by means of intermolecular distance maps. Some differences in the structures of ferro- and ferricytochrome c may exist on the surface of the molecules.     

The amino acid sequence of cytochrome c from Helix aspersa Müller (garden snail)

The amino acid sequence of a snail cytochrome c has been determined. The molecule consists of a single polypeptide chain of 104 residues, and is homologous with other mitochondrial cytochromes c. Unlike the cytochromes c from vertebrates, there is no acetyl blocking group at the N-terminus. A change in an otherwise invariant position has been observed in position 87. Comparison with amino acid sequences of cytochromes c from other sources indicates that the point of divergence of the molluscs and the vertebrates in evolutionary time was 720 million years ago. Experimental details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50009 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972), 126, 5.