Purification of a hexaheme cytochrome c552 from Escherichia coli K 12 and its properties as a nitrite reductase

Anaerobic cytochrome c552 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a mutant of Escherichia coli K 12 that synthesizes an increased amount of this pigment. Several molecular and enzymatic properties of the cytochrome were investigated. Its relative molecular mass was determined to be 69 000 by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. It was found to be an acidic protein that existed in the monomeric form in the native state. From its heme and iron contents, it was concluded to be a hexaheme protein containing six moles of heme c/mole protein. The amino-acid composition and other properties of the purified cytochrome c552 indicated its similarity to Desulfovibrio desulfuricans hexaheme cytochrome. The cytochrome c552 showed nitrite and hydroxylamine reductase activities with benzyl viologen as an artificial electron donor. It catalyzed the reduction of nitrite to ammonia in a six-electron transfer. FMN and FAD also served as electron donors for the nitrite reduction. The apparent Michaelis constants for nitrite and hydroxylamine were 110 microM and 18 mM, respectively. The nitrite reductase activity of the cytochrome c552 was inhibited effectively by cupric ion and cyanide.     

Cytochrome c oxidase in prokaryotes

Abstract Several heme aa ₃-type cytochrome c oxidases, purified from the cytoplasmic membranes of bacteria, are able to catalyze the same reactions as the structurally far more complex eukaryotic enzyme, i.e., electron transport from cytochrome c to oxygen coupled to proton translocation. However, these oxidases show a very simple subunit pattern, and moreover, individual polypeptides even have homologous amino-acid sequences. This review summarizes the present data on purified bacterial cytochrome c oxidases and relates these findings to results obtained with the mitochondrial enzymes.     

The effect of cytochrome c and its `dimer'' on electron transfer and energy transformation

A preparation that contained cytochrome c, mainly in the form of its ;dimer', was studied and compared with native cytochrome c with respect to its ability to support electron transfer and energy transformation in cytochrome c-depleted rat liver mitochondria. When the depleted mitochondria were titrated with either cytochrome c or the ;dimer', the extent of coupling between respiration and phosphorylation was enhanced, as manifested by an increase in the P/O ratio. The ;dimer' was relatively ineffective as an electron carrier in the respiratory system, but it was as effective as cytochrome c in reconstitution of oxidative phosphorylation in depleted mitochondria. Addition of ;dimer' to the depleted mitochondria, in the presence of a low, non-saturating concentration of cytochrome c, increased the P/O ratio without concomitant stimulation of respiration. Both cytochrome c and the ;dimer' stimulated spontaneous swelling and electron transport-driven proton translocation in depleted mitochondria. The pattern of action of cytochrome c and its ;dimer' is in accord with the assumption that they affect an early step in energy conservation.     

Detection, isolation and some properties of membrane proteinases from yeast mitochondria

The complete amino-acid sequences of two isocytochromes c (larval- and adult-type cytochromes c) purified from the housefly Musca domestica L. were determined. Their sequences differed at six positions from each other. More than 90% of the total cytochrome c was larval-type during larval stages. The amount of adult-type cytochrome c increased rapidly from 1 day before adult emergence, making the total cytochrome c content increase to approximately 2.5-times as much cytochrome c content as in larvae. Although the major cytochrome c species in flight muscles was adult-type, imaginal disks contained mainly larval-type cytochrome c.     

Resonance Raman spectroscopy indicates a lysine as the sixth iron ligand in cytochrome f

The resonance Raman spectrum of turnip cytochrome f is similar to that of other c-type cytochromes with the exception of a single band at 1532 cm-1 which is shifted to lower frequency relative to its position (1542-1545 cm-1) in other c-type cytochromes. Comparison of the frequency of this band with that in alkylated cytochrome c at high pH suggests that the sixth heme iron ligand in cytochrome f is a deprotonated lysine amino group rather than a methionine sulfur. Comparison of the amino-acid sequences of cytochromes f and c1 suggests lysine-145 as a likely candidate for the sixth heme iron ligand in cytochrome f.     

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.     

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

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

The role of cytochrome c diffusion in mitochondrial electron transport

We have compared the modes and rates of cytochrome c diffusion to the rates of cytochrome c-mediated electron transport in isolated inner membranes and in whole intact mitochondria. For inner membranes, an increasing ionic strength results in an increasing rate of cytochrome c diffusion, a decreasing concentration (affinity) of cytochrome c near the membrane surface as well as near its redox partners, and an increasing rate of electron transport. For intact mitochondria, an increasing ionic strength results in a parallel, increasing rate of cytochrome c-mediated electron transport. In both inner membranes and intact mitochondria the rate of cytochrome c-mediated electron transport is highest at physiological ionic strength (100-150 mM), where the diffusion rate of cytochrome c is highest and its diffusion mode is three-dimensional. In intact mitochondria, succinate and duroquinol-driven reduction of endogenous cytochrome c is greater than 95% at all ionic strengths, indicating that cytochrome c functions as a common pool irrespective of its diffusion mode. Using a new treatment to obtain bimolecular diffusion-controlled collision frequencies in a heterogenous diffusion system, where cytochrome c diffuses laterally, pseudo-laterally, or three-dimensionally while its redox partners diffuse laterally, we determined a high degree of collision efficiency (turnover/collisions) for cytochrome c with its redox partners for all diffusion modes of cytochrome c. At physiological ionic strength, the rapid diffusion of cytochrome c in three dimensions and its low concentration (affinity) near the surface of the inner membrane mediate the highest rate of electron transport through maximum collision efficiencies. These data reveal that the diffusion rate and concentration of cytochrome c near the surface of the inner membrane are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.     

Existence of multiple forms of cytochrome P-450scc purified from bovine adrenocortical mitochondria

Three fractions of cytochrome P-450scc (denoted as fractions a, b, and c) were purified by a new procedure from bovine adrenocortical mitochondria. The amino-acid content analyses of these three fractions showed no difference. NH2-terminal amino-acid sequences of cytochrome P-450scc fractions, a and b agreed completely with the sequence deduced by nucleotide sequence of cDNA of cytochrome P-450scc mRNA (Morohashi, K., Fujii-Kuriyama, Y., Okada, Y., Sogawa, K., Hirose, T., Inayama, S. and Omura, T. (1984) Proc. Natl. Acad. Sci. USA 81, 4647-4651), whereas the sequence of fraction c showed a missing of isoleucine at the NH2-terminal. COOH-terminal ámino-acid sequences of fractions a, b and c were -Gln-Ala-COOH, identical with the deduced sequence from the cDNA. Measurements of the enzymatic activities of cholesterol side-chain cleavage reaction revealed no distinct difference among these three fractions. Although each of these fractions appeared as a single protein staining band upon SDS-polyacrylamide gel electrophoresis, these fractions showed heterogeneities upon two-dimensional electrophoresis and chromatofocusing. Fraction a contained the major form of cytochrome P-450scc, and its isoelectric point was estimated to be pH 7.8 by isoelectric focusing under both native and denatured conditions, and this value was confirmed by chromatofocusing. Neither of the carbohydrate-specific stainings (such as periodic acid-Schiff staining and lectin-peroxidase stainings using concanavalin A, wheat-germ agglutinin, and soybean agglutinin) of purified cytochrome P-450scc fractions after the electrophoretic resolution on SDS-polyacrylamide gel could show cytochrome P-450scc fractions as glycoproteins, suggesting that the heterogeneities were not due to the glycosylation state.     

Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.     

The role of blood platelets in tumor angiogenesis

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c?with low- and high-midpoint potentials, and cytochrome c? as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c-type cytochrome that has not been detected in wild-type cells. This cytochrome c has a redox midpoint potential of about +280 mV and could function as an electron donor to the photochemical reaction center in vitro. The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c?of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd?-type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c? in this bacterium is NirM. The two other cytochromes c?, of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.     

Purification and characterization of a soluble cytochrome c capable of delivering electrons to chlorate reductase in Ideonella dechloratans

The electron donor for periplasmic chlorate reductase of Ideonella dechloratans has been suggested to be a soluble cytochrome c. We describe here the purification of the 9-kDa periplasmic cytochrome c, denoted cytochrome c-Id1, and demonstrate its ability to serve as an electron donor for purified chlorate reductase. The reaction rate was found to be linearly dependent on the cytochrome c concentration in the range of 0.6-4 μM. A route for electron transport involving a soluble cytochrome c is similar to that found for other periplasmic oxidoreductases of the dimethyl sulfoxide reductase family, but different from that suggested for the (per)chlorate reductase of Dechloromonas species.     

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively.Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.     

Deuterium isotope effect of proton pumping in cytochrome c oxidase

In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron-proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3-->F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3-->O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.     

The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.

The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor.     

Reorientation of cytochrome c2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer

The reaction of water-soluble cytochrome c (c(2)) with its physiological redox partners is facilitated by electrostatic attractions between the two protein surfaces. Using spin-labeled cytochrome c(2) from Rhodobacter capsulatus and pulse electron paramagnetic resonance (EPR) measurements we compared spatial orientation of cytochrome c(2) upon its binding to surfaces of opposite charge. We observed that cytochrome c(2) can use its negatively charged "back" side when exposed to interact with positively charged surfaces (DEAE resin) which is the opposite to the use of its positively charged "front" side in physiological interaction with negatively charged binding domain of cytochrome bc(1). The later orientation is also adopted upon non-physiological binding of cytochrome c(2) to negatively charged carboxymethyl cellulose resin. These results directly demonstrate how the electric dipolar nature of cytochrome c(2) influences its orientation in interactions with charged surfaces, which may facilitate collisions with other redox proteins in a proper orientation to support physiologically-competent electron transfer. Saturation recovery EPR provides an attractive tool for monitoring spatial orientation of proteins in their interaction with surfaces in liquid phase. It is particularly valuable for metalloproteins engaged in redox reactions as a means to monitor the geometry and dynamics of formation of protein complexes in measurements that are independent of electron transfer processes.     

Interactions of cytochrome c with mitochondrial membranes. Binding to succinate-cytochrome c reductase

Methyl-4-azidobenzoimidate was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c bound to cytochrome c-depleted mitochondria with the same Kd as native cytochrome c and restored oxygen uptake to the same extent. Irradiation of cytochrome c-depleted mitochondrial membranes with 3- to 4-fold excess of photoaffinity-labeled cytochrome c over cytochrome c oxidase resulted in covalent binding of the derivative to the membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on an agarose Bio-Gel-A-5m showed that the labeled cytochrome c was bound covalently to succinate-cytochrome c reductase. The covalently bound cytochrome c was active in mediating electron transfer between its reductase and oxidase. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the succinate-cytochrome c reductase containing photoaffinity-labeled 125I-cytochrome c showed that the reductase contained a protein binding site for cytochrome c. It is suggested that cytochrome c1 is the most likely site for the cytochrome c binding in mitochondria in situ.     

Physiological role of glucoside 3-dehydrogenase and cytochrome c551 in the sugar oxidizing system of Flavobacterium saccharophilum

Flavobacterium saccharophilum cytoplasmic membranes contain several cytochromes linked to the respiratory chain. The presence of c-type cytochrome, cytochrome o, and a small amount of a-type cytochrome was proved. Cytochrome c551 was purified to electrophoretic homogeneity by ion-exchange chromatography and gel filtration from a membrane fraction of F. saccharophilum and its properties determined. Cytochrome c551 possessed absorption peaks at 407 nm in the oxidized form, and at 415, 521, 551 nm in the reduced form. The cytochrome c551 had a molecular weight of 15,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Glucoside 3-dehydrogenase of F. saccharophilum reduced the cytochrome c551 with methyl-alpha-D-glucoside, D-glucose, sucrose, or validoxylamine A. When the purified glucoside 3-dehydrogenase was incubated with methyl-alpha-D-glucoside and purified ferricytochrome c551, methyl-alpha-D-3-ketoglucoside was formed as indicated by GC-MS analysis. The addition of a substrate to the membrane fraction caused an increase in the rate of oxygen uptake and an abrupt reduction in cytochrome c551. The electron transfer in the 3-keto sugar forming system may be as follows: sugars----glucoside 3-dehydrogenase----cytochrome c551----cytochrome oxidase----O2. Thus, the electron acceptor of glucoside 3-dehydrogenase is possibly connected to the membrane-bound cytochrome system.     

Cytochromes c550, c552, and c1 in the Electron Transport Network of Paracoccus denitrificans: Redundant or Subtly Different in Function?

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c(550), c(552), or c(1) and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc(1) complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c(550)-deficient mutant did denitrify provided copper was present. The cytochrome c(552) mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc(1) complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c(550) and c(552) and to the cbb(3)-type oxidase. Cytochrome c(552) is an electron acceptor both of the cytochrome bc(1) complex and of amicyanin, as well as a dedicated electron donor to the aa(3)-type oxidase. Cytochrome c(550) can accept electrons from the cytochrome bc(1) complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c-type cytochromes also affected the concentrations of remaining cytochromes c, suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.     

Electron transfer from excited tryptophan to cytochrome c: mechanism of phosphorescence quenching?

Parvalbumin, aldolase and liver alcohol dehydrogenase (ADH), proteins exhibiting long-lived phosphorescence lifetimes at room temperature, were examined for their reactivity with ferricytochrome c (cytochrome c Fe3+) as an external electron acceptor. Illumination of a reaction mixture containing protein and cytochrome c in the absence of oxygen brought about reduction of cytochrome c in relation to the duration of light. The largest portion of reduced cytochrome c was found with a sample containing ADH, where a 50% reduction of cytochrome c was reached after 5 min of illumination with a xenon lamp. Parvalbumin and aldolase were about half as effective under the same conditions. Several lines of evidence support the idea that the reaction of cytochrome c occurred by a long-range electron transfer from the excited triplet state of tryptophan. First, cytochrome c quenches the tryptophan phosphorescence and with parvalbumin, its bimolecular quenching rate constant, kq, was 2.9 x 10(6) M-1 s-1. Second, when the illuminated reaction mixture was supplied with 0.2 mM to 1 mM nitrite, a concentration range of nitrite which quenches the tryptophan phosphorescence but not the fluorescence, the amount of reduced cytochrome c on illumination markedly decreased. Finally, for all illuminated protein samples, the extent of cytochrome c reduction occurred parallel to a decrease in tryptophan content as judged from a decrease in fluorescence intensity and/or a decrease in tryptophan absorption at 280 nm.