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PubMed1975

Cytochrome c interaction with membranes. Absorption and emission spectra and binding characteristics of iron-free cytochrome c.

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state.

Animals↗
PubMed1983

Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1. Characterization of fluorescent Zn(II)-substituted cytochrome c.

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.

Ascomycota↗
PubMed2005

Unusual dehydroxylation of antimicrobial amidoxime prodrugs by cytochrome b5 and NADH cytochrome b5 reductase.

Furamidine is an effective antimicrobial agent; however, oral potency of furamidine is poor. A prodrug of furamidine, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime (DB289), has greatly improved oral potency. DB289 is transformed to furamidine via O-demethylation, and N-dehydroxylation reactions with four intermediate metabolites formed. The O-demethylation reactions have been shown to be catalyzed by cytochrome P450. The enzymes catalyzing the reductive N-dehydroxylation reactions have not been determined. The objective of this study was to identify the enzymes that catalyze N-dehydroxylation of metabolites M1, a monoamidoxime, and M2, a diamidoxime, formed during generation of furamidine. M1 and M2 metabolism was investigated using human liver microsomes and human soluble cytochrome b5 and NAD cytochrome b5 reductase, expressed in Escherichia coli. Kinetics of M1 and M2 reduction by human liver microsomes exhibited high affinity and moderate capacity. Metabolism was significantly inhibited by antibodies to cytochrome b5 and b5 reductase and by chemical inhibitors of b5 reductase. The amidoximes were efficiently metabolized by liver mitochondria, which contain cytochrome b5/b5 reductase, but not by liver cytosol, which contains minimal amounts of these proteins. Expressed cytochrome b5/b5 reductase, in the absence of any other proteins, efficiently catalyzed reduction of both amidoximes. K(m) values were similar to those for microsomes, and V(max) values were 33- to 36-fold higher in the recombinant system compared with microsomes. Minimal activity was seen with cytochrome b5 or b5 reductase alone or with cytochrome P450 reductase alone or with cytochrome b5. These results indicate that cytochrome b5 and b5 reductase play a direct role in metabolic activation of DB289 to furamidine.

Benzamidines↗
PubMed2004

NADH cytochrome b5 reductase and cytochrome b5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans.

Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b5 reductase and cytochrome b5 were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b5 reductase and cytochrome b5, expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent Km values similar to those found in human liver microsomes and specific activities (Vmax) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b5 in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b5 reductase and cytochrome b5 significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b5 reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b5 reductase and cytochrome b5 in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.

Amides↗
PubMed1980

Cytochrome b reducible by succinate in an isolated succinate dehydrogenase-cytochrome b complex from Bacillus subtilis membranes.

In previous work with membranes of Bacillus subtilis, the succinate dehydrogenase complex was isolated by immunoprecipitation of Triton X-100-solubilized membranes. The complex included a polypeptide with an apparent molecular weight of 19,000, probably attributable to apocytochrome. This paper reports the further characterization of this cytochrome and its relation to the respiratory chain of B. subtilis. The cytochrome was identified as cytochrome b, and its difference absorption spectra showed maxima at 426, 529, and 558 nm at room temperature. The oxidized cytochrome had an absorption maximum at 413 nm. The cytochrome was reduced by succinate in the isolated succinate dehydrogenase complex and in Triton X-100-solubilized membranes. In whole membranes cytochromes b, c, and a were reduced by succinate. In membranes from a mutant containing normal cytochromes but lacking succinate dehydrogenase no reduction of cytochrome was seen with succinate. It was concluded that the isolated succinate dehydrogenase-cytochrome b complex is a functional unit in the intact B. subtilis membrane. An accompanying paper describes cytochrome b as a structural unit involved in the membrane binding of succinate dehydrogenase.

Bacillus subtilis↗
PubMed2003

A mutant of Paracoccus denitrificans with disrupted genes coding for cytochrome c550 and pseudoazurin establishes these two proteins as the in vivo electron donors to cytochrome cd1 nitrite reductase.

In Paracoccus denitrificans, electrons pass from the membrane-bound cytochrome bc(1) complex to the periplasmic nitrite reductase, cytochrome cd(1). The periplasmic protein cytochrome c(550) has often been implicated in this electron transfer, but its absence, as a consequence of mutation, has previously been shown to result in almost no attenuation in the ability of the nitrite reductase to function in intact cells. Here, the hypothesis that cytochrome c(550) and pseudoazurin are alternative electron carriers from the cytochrome bc(1) complex to the nitrite reductase was tested by construction of mutants of P. denitrificans that are deficient in either pseudoazurin or both pseudoazurin and cytochrome c(550). The latter organism, but not the former (which is almost indistinguishable in this respect from the wild type), grows poorly under anaerobic conditions with nitrate as an added electron acceptor and accumulates nitrite in the medium. Growth under aerobic conditions with either succinate or methanol as the carbon source is not significantly affected in mutants lacking either pseudoazurin or cytochrome c(550) or both these proteins. We concluded that pseudoazurin and cytochrome c(550) are the alternative electron mediator proteins between the cytochrome bc(1) complex and the cytochrome cd(1)-type nitrite reductase. We also concluded that expression of pseudoazurin is mainly controlled by the transcriptional activator FnrP.

Anaerobiosis↗
PubMed1989

Identification of the cytochrome P-450 isozymes responsible for testosterone oxidation in rat lung, kidney, and testis: evidence that cytochrome P-450a (P450IIA1) is the physiologically important testosterone 7 alpha-hydroxylase in rat testis.

Previous studies have shown that several forms of cytochrome P-450 present in rat liver microsomes oxidize testosterone with a high degree of regio- and stereospecificity. The aim of this study was to characterize the pathways of testosterone oxidation catalyzed by rat extrahepatic microsomes. Lung, kidney, testis, prostate, and brain were isolated from 3- and 14-week-old-male Sprague-Dawley rats. Microsomes from lung, kidney, and testis catalyzed distinctly different pathways of testosterone oxidation, whereas microsomes from prostate and brain failed to hydroxylate testosterone directly in a time- and protein-dependent manner. Lung microsomes from immature and mature rats converted testosterone to 16 alpha-hydroxytestosterone, 16 beta-hydroxytestosterone, and androstenedione. Lung microsomes were shown by Western immunoblot to contain cytochrome P-450b (P450IIB1), which has been shown previously to catalyze these three pathways of testosterone oxidation. Antibody against cytochrome P-450b strongly inhibited (greater than 80%) androstenedione formation and completely inhibited (greater than 95%) the 16 alpha- and 16 beta-hydroxylation of testosterone catalyzed by lung microsomes (as did carbon monoxide and antibody against NADPH-cytochrome P-450 reductase). Kidney microsomes from mature male rats converted testosterone to 2 alpha-hydroxytestosterone, 16 alpha-hydroxytestosterone, and androstenedione, whereas only the latter pathway was catalyzed by kidney microsomes from immature rats. Kidney microsomes from mature male rats were shown by Western immunoblot to contain cytochrome P-450h (P450IIC11), which has been shown previously to convert testosterone to 2 alpha-hydroxytestosterone, 16 alpha-hydroxytestosterone, and androstenedione. Antibody against cytochrome P-450h completely inhibited (greater than 95%) the 2 alpha- and 16 alpha-hydroxylation of testosterone by kidney microsomes, but had little effect on androstenedione formation, which is catalyzed by 17 beta-hydroxysteroid dehydrogenase. Testicular microsomes from mature, but not immature, rats catalyzed the 7 alpha-hydroxylation of testosterone. Previous studies have shown that this reaction is catalyzed in liver microsomes by cytochrome P-450a (P450IIA1). Testicular microsomes from mature, but not immature, rats were shown by Western immunoblot to contain cytochrome P-450a. Antibody against cytochrome P-450a or NADPH-cytochrome P-450 reductase completely inhibited (greater than 95%) the 7 alpha-hydroxylation of testosterone by testicular microsomes. A 90:10 atmosphere of carbon monoxide and oxygen did not appreciably block the 7 alpha-hydroxylation of testosterone by testicular microsomes, wh

Animals↗
PubMed1992

Zone-specific expression of aldosterone synthase cytochrome P-450 and cytochrome P-45011 beta in rat adrenal cortex: histochemical basis for the functional zonation.

Zonal distribution of aldosterone synthase cytochrome P-450 and cytochrome P-45011 beta in rat adrenocortex was investigated immunochemically using specific antibodies to these enzymes. Localization of aldosterone synthase cytochrome P-450 (cytochrome P-450aldo), a recently identified enzyme that converts deoxycorticosterone to aldosterone in rat adrenocortex was strictly confined to two or three outermost cell layers in the zona glomerulosa. In contrast, cytochrome P-45011 beta, which forms corticosterone, but not aldosterone, from deoxycorticosterone, was localized in the zona fasciculata-reticularis and not in the zona glomerulosa. Neither enzyme was detected in the medulla or the capsule. The functional zonation of adrenocortex with respect to aldosterone and corticosterone syntheses is, thus, ascribable to the localization of cytochromes P-450aldo and P-45011 beta in the respective zones. When rats were maintained under Na-depleted conditions for 10 days, the zona glomerulosa cells containing cytochrome P-450aldo proliferated to 10-15 layers, the thickness of which was 5-7-fold that in the nonstimulated rats. Proliferation of the cytochrome P-450aldo-positive cells into the zona fasciculata-reticularis was also observed along with arterial walls. Under these conditions, no significant change in the distribution of cytochrome P-45011 beta was noted. These results indicate that the angiotensin-II stimuli, which had been elicited by the low Na treatment, promoted proliferation of the glomerulosa cells, resulting in increased expression of cytochrome P-450aldo in rat adrenocortex.

Adrenal Cortex↗
PubMed2001

Solution structure of cytochrome b(5) mutant (E44/48/56A/D60A) and its interaction with cytochrome c.

Using 1617 meaningful NOEs with 188 pseudocontact shifts, a family of 35 conformers of oxidized bovine microsomal cytochrome b5 mutant (E44/48/56A/D60A) has been obtained and is characterized by good resolution (rmsd to the mean structure are 0.047 +/- 0.007 nm and 0.095 +/- 0.008 nm for backbone and heavy atoms, respectively). The solution structure of the mutant, when compared with the X-ray structure of wild-type cytochrome b(5), has no significant changes in the whole folding and secondary structure. The binding between cytochrome b(5) and cytochrome c shows that the association constant of the mutant-cytochrome c complex is much lower than the one for wild-type complex (2.2 x 10(4) M(-1) vs. 5.1 x 10(3) M(-1)). The result suggests the four acidic residues have substantial effects on the formation of the complex between cytochrome b(5) and cytochrome c, and therefore it is concluded reasonably that the electrostatic interaction plays an important role in maintaining the stability and specificity of the complex formed. The competition between the ferricytochrome b(5) mutant and [Cr(oxalate)(3)](3-) for ferricytochrome c shows that site III of cytochrome c, which is a strong binding site to wild-type cytochrome b(5), still binds to the mutant with relatively weaker strength. Our results indicate that certain bonding geometries do occur in the interaction between the present mutant and cytochrome c and these geometries, which should be quite different from the ones of the Salemme and Northrup models.

Amino Acid Sequence↗
PubMed1977

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.

Animals↗
PubMed1990

[Benzodiazepines as cytochrome P-450 and cytochrome b5 inductors in rat liver microsomes].

Among the benzodiazepines tested (diazepam, oxazepam, clonazepam, nitrazepam and chlordiazepoxide ) chlordiazepoxide is the most potent inducer of cytochrome P-450, diazepam is a poor inducer, whereas clonazepam and nitrazepam do not possess a capacity to induce of cytochrome P-450. On the other hand, microsomal cytochrome b5 is induced by diazepam only. The extent of induction of cytochrome P-450 and cytochrome b5 depends on the environmental temperature. Chlordiazepoxide is the most potent inducer of cytochrome P-450 in rats exposed to an ambient temperature of 28 degrees C, whereas diazepam have the highest induction ability in rats exposed to 35 degrees C. On the other hand, nitrazepam increased the content of cytochrome b5 in rats exposed to temperature of 35 degrees C only; diazepam was the most potent inducer of cytochrome b5 in rats exposed to temperature of 21 degrees C. These results indicate that high ambient temperature is a factor modifying the ability of benzodiazepines to induce of microsomal cytochrome P-450 and cytochrome b5.

Animals↗
PubMed1985

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c.

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.

Animals↗
PubMed1993

Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c1.

The reactions of a beef heart cytochrome c1 preparation containing the hinge protein with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) (Ru(II)) were studied by flash photolysis. All of the ruthenium-cytochrome c derivatives formed complexes with cytochrome c1 in low ionic strength buffer (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a 0.4-microseconds laser flash resulted in rapid electron transfer to the ferric heme group in cytochrome c, followed by electron transfer from the ferrous heme group of cytochrome c to the ferric heme group of cytochrome c1. The kinetic difference spectra displayed maxima at 546 nm and minima at 554 nm characteristic of electron transfer between the two cytochromes. The rate constants were independent of concentration at low ionic strength, indicating intracomplex electron transfer. The rate constants were 4,800, 6,800, 22,000, and 22,000 s-1 for cytochrome c derivatives modified at lysines 13, 27, 25, and 72, respectively. The observed rate constants were independent of ionic strength up to about 50 nM and then decreased progressively with further increases in ionic strength indicating dissociation of the complex. Second-order kinetics were observed at 310 mM ionic strength, with rate constants of 1.0 x 10(6), 1.6 x 10(7), 1.2 x 10(8), and 3.0 x 10(7) M-1 s-1 for the derivatives modified at lysines 13, 27, 25, and 72, respectively. The ionic strength dependence of the second-order rate constants is comparable to that involving native horse cytochrome c and is consistent with electron transfer reactions between oppositely charged proteins.

Animals↗
PubMed1981

The triphasic reduction of cytochrome b in the succinate-cytochrome c reductase.

In the succinate-cytochrome c reductase, the reduction of cytochrome b has been found to be triphasic: an initial rapid partial reduction was followed first by a rapid oxidation and then finally by a slow reduction. The initial reduction of cytochrome b was faster than that of cytochrome c1 and the final slow reduction of cytochrome b began when cytochrome c1 reduction was approaching completion. In presence of the inhibitors antimycin A or HQNO the reduction of cytochrome b became monophasic. Hysteresis or a kinetic cooperative effect of a factor controlling cytochrome b oxidation has been suggested as a possible explanation for the triphasic reduction of cytochrome b.

Animals↗
PubMed1985

Palmitoyl-CoA elongation in brain microsomes: dependence on cytochrome b5 and NADH-cytochrome b5 reductase.

Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.

4-Chloromercuribenzenesulfonate↗
PubMed1984

Cytochrome electron spin resonance line shapes, ligand fields, and components stoichiometry in ubiquinol-cytochrome c oxidoreductase.

The EPR spectra of the cytochromes in ubiquinol-cytochrome c oxidoreductase (Complex III) have peaks at g = 3.78 (cytochrome b566) g = 3.45 (cytochrome b562) and g = 3.35 (cytochrome c1). The highly asymmetric peak of cytochrome b566 has been simulated using an arbitrary gaussian distribution of crystal field parameters. The asymmetry is due to the nonlinear relationship between field position and crystal field. The results suggest that the b cytochromes have bis-imidazole ligation. The gz peak of cytochrome c1 was also found to be asymmetric; simulations suggest histidine-methionine ligation. No other important cytochrome components were needed to simulate the spectrum of the oxidized complex; these results are consistent with 1:1:1 stoichiometry of components. These results argue against any asymmetric dimer model for Complex III.

Cytochrome b Group↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2001

Molecular analysis of the cytochrome bc1-aa3 branch of the Corynebacterium glutamicum respiratory chain containing an unusual diheme cytochrome c1.

In this work, the genes for cytochrome aa3 oxidase and the cytochrome bc1 complex in the gram-positive soil bacterium Corynebacterium glutamicum were identified. The monocistronic ctaD gene encoded a 65-kDa protein with all features typical for subunit I of cytochrome aa3 oxidases. A ctaD deletion mutant lacked the characteristic 600 nm peak in redox difference spectra, and growth in glucose minimal medium was strongly impaired. The genes encoding subunit III of cytochrome aa3 (ctaE) and the three characteristic subunits of the cytochrome bc1 complex (qcrABC) were clustered in the order ctaE-qcrCAB. Analysis of the deduced primary structures revealed a number of unusual features: (1) cytochrome c1 (QcrC, 30 kDa) contained two Cys-X-X-Cys-His motifs for covalent heme attachment, indicating that it is a diheme c-type cytochrome; (2) the 'Rieske' iron-sulphur protein (QcrA, 45 kDa) contained three putative transmembrane helices in the N-terminal region rather than only one; and (3) cytochrome b (QcrB, 60 kDa) contained, in addition to the conserved part with eight transmembrane helices, a C-terminal extension of about 120 amino acids, which presumably is located in the cytoplasm. Staining of C. glutamicum proteins for covalently bound heme indicated the presence of a single, membrane-bound c-type cytochrome with an apparent molecular mass of about 31 kDa. Since this protein was missing in a qcrCAB deletion mutant, it most likely corresponds to cytochrome c1. Similar to the deltactaD mutant, the deltaqcrCAB mutant showed strongly impaired growth in glucose minimal medium, which indicates that the bc1-aa3 pathway is the main route of respiration under these conditions.

Amino Acid Sequence↗