Characterization of recombinant horse cytochrome c synthesized with the assistance of Escherichia coli cytochrome c maturation factors

Cytochromes c are characterized by the presence of a protoporphyrin IX group covalently attached to the polypeptide via one or two thioether bonds to Cys side chains. The heme attachment process, known as cytochrome c maturation, occurs posttranslationally in the periplasm (for bacterial cytochromes c) or in the mitochondrial intermembrane space (for eukaryotic cytochromes c) through a pathway dependent on the organism. It is demonstrated in this work that a mitochondrial cytochrome c expressed in Escherichia coli that undergoes maturation under control of the E. coli cytochrome c maturation factors achieves a native-like structure and stability. The recombinant protein is characterized spectroscopically (by circular dichroism (CD), absorption, and nuclear magnetic resonance (NMR) spectroscopy) and it is verified that the heme and its environment are indistinguishable from authentic horse cytochrome c. Mass spectrometry reveals that the recombinant protein is not acetylated at the N terminus, however, no significant effect on protein structure or stability is detected as a result.     

Some recent advances relating to prokaryotic cytochrome c reductases and cytochrome c oxidases.

Prokaryotic systems provide excellent experimental opportunities for exploring structure/function relationships for the complex, membrane-bound, multisubunit enzymes responsible for the reduction and subsequent oxidation of c-type cytochromes in respiratory or photosynthetic electron transport chains. Two points are made in this mini-review: (1) The eukaryotic and prokaryotic aa3-type cytochrome c oxidases are members of an apparently large superfamily of structurally related respiratory oxidases. This superfamily displays considerable variation in terms of the heme prosthetic groups (a or b) as well as the substrate oxidized (quinol or cytochrome c). The relationships among these enzymes help to facilitate explorations of how they work. (2) Molecular biology techniques can be used to generate intact, redox-active, water-soluble domains of membrane-bound subunits. These soluble domains can be used for detailed examination, including obtaining high resolution structure by NMR techniques or by X-ray crystallography. This approach is being used to study the soluble heme-binding domain of cytochrome c1 from the bc1 complex of Rhodobacter sphaeroides.     

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c3 from Desulfovibrio vulgaris Hildenborough: kinetic, microcalorimetric, EPR and electrochemical studies

The complex formation between the tetraheme cytochrome c3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c3 and Hmc demonstrates a redox-shuttling role of cytochrome c3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed. A model with cytochrome c3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.     

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.     

Comparison of low oxidoreduction potential cytochrome c553 from Desulfovibrio vulgaris with the class I cytochrome c family

The cytochrome c553 from Desulfovibrio vulgaris (DvH c553) is of importance in the understanding of the relationship of structure and function of cytochrome c due to its lack of sequence homology with other cytochromes, and its abnormally low oxido-reduction potential. In evolutionary terms, this protein also represents an important reference point for the understanding of both bacterial and mitochondrial cytochromes c. Using the recently determined nuclear magnetic resonance (NMR) structure of the reduced protein we compare the structural, dynamic, and functional characteristics of DvH c553 with members of both the mitochondrial and bacterial cytochromes c to characterize the protein in the context of the cytochrome c family, and to understand better the control of oxido-reduction potential in electron transfer proteins. Despite the low sequence homology, striking structural similarities between this protein and representatives of both eukaryotic [cytochrome c from tuna (tuna c)] and prokaryotic [Pseudomonas aeruginosa c551 (Psa c551)] cytochromes c have been recognized. The previously observed helical core is also found in the DvH c553. The structural framework and hydrogen bonding network of the DvH c553 is most similar to that of the tuna c, with the exception of an insertion loop of 24 residues closing the heme pocket and protecting the propionates, which is absent in the DvH c553. In contrast, the Psa c551 protects the propionates from the solvent principally by extending the methionine ligand arm. The electrostatic distribution at the recognized encounter surface around the heme in the mitochondrial cytochrome is reproduced in the DvH c553, and corresponding hydrogen bonding networks, particularly in the vicinity of the heme cleft, exist in both molecules. Thus, although the cytochrome DvH c553 exhibits higher primary sequence homology to other bacterial cytochromes c, the structural and physical homology is significantly greater with respect to the mitochondrial cytochrome c. The major structural and functional difference is the absence of solvent protection for the heme, differentiating this cytochrome from both reference cytochromes, which have evolved different mechanisms to cover the propionates. This suggests that the abnormal redox potential of the DvH c553 is linked to the raised accessibility of the heme and supports the theory that redox potential in cytochromes is controlled by heme propionate solvent accessibility.     

MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association.

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely.The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit.The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.     

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.     

Amino acid sequence of cytochrome c from rice

The amino acid sequence of cytochrome c purified from rice, Oryza sativa L., was determined. The complete amino acid sequence of rice cytochrome c is as follows: Ac-Ala-8-Ser-Phe-Ser-Glu-Ala-Pro-Pro-Gly1-Asn-Pro-Lys-Ala-Gly-Glu-Lys-Ile-Phe10-Lys-Thr-Lys-Cys-Ala-Glx-Cys-His-Thr-Val20-Asp-Lys-Gly-Ala-Gly-His-Lys-Glx-Gly-Pro30-Asx-Leu-Asx-Gly-Leu-Phe-Gly-Arg-Glx-Ser40-Gly-Thr-Thr-Pro-Gly-Tyr-Ser-Tyr-Ser-Thr50-Ala-Asp-Lys-Asn-Met-Ala-Val-Ile-Trp-Glx60-Glx-Asx-Thr-Leu-Tyr-Asp-Tyr-Leu-Leu-Asn70-Pro-TML-Lys-Tyr-Ile-Pro-Gly-Thr-Lys-Met80-Val-Phe-Pro-Gly-Leu-TML-Lys-Pro-Glx-Glx90-Arg-Ala-Asp-Leu-Ile-Ser-Tyr-Leu-Lys-Glu100-Ala-Thr-Ser (Ac = acetyl group, TML = epsilon-N-trimethyllsine). The primary structure of rice cytochrome c was found to be homologous with those of other plant cytochromes c reported so far; it possesses general features common to plant cytochromes c, and all the invariant residues characterized in dicotyledonous cytochromes c are also conserved in the sequence of rice cytochrome c, as well as those of other monocotyledonous cytochromes c. The distinctive features of rice cytochrome c are a high content of proline residues, their unique locations in the sequence and the presence of a serine residue at position 96.     

Change in the ratio of cytochrome oxidase activity to nitrite reductase activity of Pseudomonas aeruginosa nitrite reductase with the kind of C-type cytochrome used as an electron donor.

The ratio between the nitrite reductase and cytochrome oxidase activities of Pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2.] varies with kind of C-type cytochrome used as the electron donor. Withe cytochrome c-548, 554 (Micrococcus sp.), the nitrite reductase activity is greater than the cytochrome oxidase activity, while the former is smaller than the latter with cytochrome c-554 (Navicula pelliculosa). The aerobic oxidation catalyzed by this enzyme of denitrifying bacterial ferrocytochrome c is greatly accelerated on addition of nitrite, while that of the algal ferrocytochrome c is not affected or is even depressed by the salt. An accelerative effect of nitrite is generally observed with many kinds of C-type cytochromes which react with the enzyme very or fairly rapidly. The difference in the ratio of the two activities of the enzyme seems to arise according to whether or not nitrite affects the interaction of C-type cytochrome with the enzyme.     

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.     

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.     

Role of the hinge protein in the electron transfer between cardiac cytochrome c1 and c. Equilibrium constants and kinetic probes

A role of the hinge protein is studied in the electron transfer reaction between cytochromes c1 and c, using highly purified "one-band" cytochrome c1 and "two-band" cytochrome c1. The results show that the hinge protein (Hp), which is essential for a stable ionic strength-sensitive c1-Hp-c complex, seems to play a certain role in electron transfer between cytochromes c1 and c; Keq for electron transfer reaction between cytochromes c1 and c in the presence of the hinge protein is found to be about 40% higher than that in the absence of the hinge protein at low ionic strength, but no difference exists at high ionic strength. We propose a hypothesis that the hinge protein may function as regulator for the electron transfer reaction between cytochromes c1 and c, and this may be at least one of the roles of the hinge protein in mitochondria.     

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.     

Amino acid sequence of Paracoccus denitrificans cytochrome c550.

The amino acid sequence of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been established by a combination of standard chemical techniques and interpretation of a 2.5 A resolution x-ray electron density map. Peptides derived from a trypsin digest were chemically sequenced, and then ordered by fitting them to the density map. The amino acid compositions of chymotryptic peptides confirmed the x-ray map ordering the tryptic peptides. The amino acid sequence of this respiratory, prokaryotic cytochrome with 134 residues is discussed in relation to those of eukaryotic respiratory cytochrome c (103 to 113 amino acids), and prokaryotic, photosynthetic c2 (103 to 124 amino acids). At the primary structure level, c and c550 differ no more from cytochromes c2 than the various cytochromes c2 do from one another. It is suggested that the respiratory electron transport chain in prokaryotes and eukaryotes is a relatively late evolutionary offshoot of the photosynthetic electron transport chain in purple non-sulfur bacteria.     

The NT-26 cytochrome c552 and its role in arsenite oxidation

Arsenite oxidation by the facultative chemolithoautotroph NT-26 involves a periplasmic arsenite oxidase. This enzyme is the first component of an electron transport chain which leads to reduction of oxygen to water and the generation of ATP. Involved in this pathway is a periplasmic c-type cytochrome that can act as an electron acceptor to the arsenite oxidase. We identified the gene that encodes this protein downstream of the arsenite oxidase genes (aroBA). This protein, a cytochrome c(552), is similar to a number of c-type cytochromes from the alpha-Proteobacteria and mitochondria. It was therefore not surprising that horse heart cytochrome c could also serve, in vitro, as an alternative electron acceptor for the arsenite oxidase. Purification and characterisation of the c(552) revealed the presence of a single heme per protein and that the heme redox potential is similar to that of mitochondrial c-type cytochromes. Expression studies revealed that synthesis of the cytochrome c gene was not dependent on arsenite as was found to be the case for expression of aroBA.     

Escherichia coli genes required for cytochrome c maturation.

The so-called aeg-46.5 region of Escherichia coli contains genes whose expression is induced under anaerobic growth conditions in the presence of nitrate or nitrite as the terminal electron acceptor. In this work, we have examined more closely several genes of this cluster, here designated ccmABCDEFGH, that are homologous to two separate Bradyrhizobium japonicum gene clusters required for the biogenesis of c-type cytochromes. A deletion mutant of E. coli which lacked all of these genes was constructed. Maturation of indigenous c-type cytochromes synthesized under anaerobic respiratory conditions, with nitrite, nitrate, or trimethylamine N-oxide as the electron acceptor, was found to be defective in the mutant. The biogenesis of foreign cytochromes, such as the soluble B. japonicum cytochrome c550 and the membrane-bound Bacillus subtilis cytochrome c550, was also investigated. None of these cytochromes was synthesized in its mature form when expressed in the mutant, as opposed to the situation in the wild type. The results suggest that the E. coli ccm gene cluster present in the aeg-46.5 region is required for a general pathway involved in cytochrome c maturation.     

Molecular cloning and nucleotide sequence of a cDNA encoding Euglena gracilis cytochrome c1

The amino acid sequence of the mature protein of Euglena gracilis cytochrome c1 was determined by sequencing of its cDNA. A cDNA expression library was constructed from Euglena poly(A)+ RNA in phage lambda gt11 and screened with an antiserum raised against cytochrome c1 polypeptide isolated from purified E. gracilis complex III. An isolated cDNA clone consisted of 872 base pairs and encoded the mature protein with 243 amino acids. The deduced amino acid sequence contained the unusual heme binding sequence-Phe-Ala-Pro-Cys-His- (Mukai, K. et al. (1989) Eur. J. Biochem. 178, 649-656) instead of the typical sequence,-Cys-X-Y-Cys-His-, commonly found in C-type cytochromes. Comparison of the sequence with those of several other cytochromes c1 revealed that Euglena cytochrome c1 conserved the residues probably ligating heme-iron, those supposed to interact with cytochrome c and regions anchoring the mitochondrial inner membrane.     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

The cytochrome c peroxidase of Paracoccus denitrificans.

The size, visible absorption spectra, nature of haem and haem content suggest that the cytochrome c peroxidase of Paracoccus denitrificans is related to that of Pseudomonas aeruginosa. However, the Paracoccus enzyme shows a preference for cytochrome c donors with a positively charged 'front surface' and in this respect resembles the cytochrome c peroxidase from Saccharomyces cerevisiae. Paracoccus cytochrome c-550 is the best electron donor tested and, in spite of an acidic isoelectric point, has a markedly asymmetric charge distribution with a strongly positive 'front face'. Mitochondrial cytochromes c have a much less pronounced charge asymmetry but are basic overall. This difference between cytochrome c-550 and mitochondrial cytochrome c may reflect subtle differences in their electron transport roles. A dendrogram of cytochrome c1 sequences shows that Rhodopseudomonas viridis is a closer relative of mitochondria than is Pa. denitrificans. Perhaps a mitochondrial-type cytochrome c peroxidase may be found in such an organism.