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PubMed1989

The cytochrome c oxidase-cytochrome c complex: spectroscopic analysis of conformational changes in the protein-protein interaction domain.

Binding to cytochrome c oxidase induces a conformational change in the cytochrome c molecule. This conformational change has been characterized by comparing the binding of native cytochrome c and chemically modified cytochrome c derivatives to bovine cytochrome c oxidase by using absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopy. The following derivatives were analyzed: (i) cytochrome c modified at all 19 lysine residues to yield the (N epsilon-acetimidyl)19 cytochrome c, (N epsilon-isopropyl)19 cytochrome c, and (N epsilon,N epsilon-dimethyl)19 cytochrome c; (ii) cytochrome c in which Met65 and Met80 are converted to the methionine sulfoxide; (iii) cytochrome c with a single break in the polypeptide chain at Arg38 or Gly37. The derivatives bind to cytochrome c oxidase at a ratio of one heme c per heme aa3. The association constants are similar to that of native cytochrome c except for (N epsilon-isopropyl)19 and (N epsilon,N epsilon-dimethyl)19 cytochromes c, which bind respectively four times and six times less strongly. The derivatives are good substrates for the cytochrome c oxidase reaction. The spectral changes accompanying the binding of the modified cytochromes c to cytochrome c oxidase are quite different from the spectral changes observed with native cytochrome c. The different optical absorption and MCD changes are explained by a polarity change around the exposed heme edge in the cytochrome c-cytochrome c oxidase complex. The CD changes indicate a conformational rearrangement restricted to the surface area surrounding the exposed heme edge. The rearrangement may involve a movement of the evolutionarily conserved Phe82 out of the vicinity of the heme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Induction of two immunochemically related rat liver cytochrome P-450 isozymes, cytochromes P-450c and P-450d, by structurally diverse xenobiotics.

We have previously shown that purified rat liver cytochromes P-450c and P-450d share some but not all immunochemical determinants (Reik, L. M., Levin, W., Ryan, D.E., and Thomas, P.E. (1982) J. Biol. Chem. 257, 3950-3957). Antibody to cytochrome P-450d cross-reacts with cytochrome P-450c to form an immunoprecipitin band in the Ouchterlony test, but no detectable immunoprecipitin ring is formed in a radial immunodiffusion assay. However, the addition of purified cytochrome P-450c to purified cytochrome P-450d in the radial immunodiffusion assay alters the cytochrome P-450d standard curve. Appropriate corrections have been made for the interference of cytochrome P-450c in the immunoquantitation of cytochrome P-450d. Twelve structurally diverse xenobiotics have been examined for their capacity to modulate the levels of cytochrome P-450d, as well as cytochromes P-450a, P-450b, and P-450c, in rat liver microsomes. Five compounds (isosafrole, 3-methylcholanthrene, beta-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and phenothiazine) and the polychlorinated biphenyl mixture Aroclor 1254 are potent in vivo inducers of cytochrome P-450d (0.44-0.89 nmol of cytochrome P-450d/mg of microsomal protein). Control rats have low levels of this microsomal hemoprotein (0.04-0.05 nmol of cytochrome P-450d/mg of microsomal protein). Isosafrole induces cytochrome P-450d to a greater extent than cytochrome P-450c, Aroclor 1254 induces both hemoproteins to similar extents, and the remaining four compounds preferentially induce cytochrome P-450c relative to cytochrome P-450d. All of these structurally diverse compounds induce cytochromes P-450d and P-450c, suggesting that the inducibility of these cytochrome P-450 isozymes, but not cytochromes P-450a and P-450b, is linked.

Animals↗
PubMed1988

Rate enhancement of the internal electron transfer in cytochrome c oxidase by the formation of a peroxide complex; its implication on the reaction mechanism of cytochrome c oxidase.

The oxidation of reduced cytochrome c oxidase by hydrogen peroxide was investigated with stopped-flow methods. It was reported by us previously (A.C.F. Gorren, H. Dekker and R. Wever (1986) Biochim. Biophys. Acta 852, 81-92) that at low H2O2 concentrations cytochrome a is oxidised simultaneously with cytochrome a3, but that at higher H2O2 concentrations the oxidation of cytochrome a is slower than that of cytochrome a3. We now report that for high peroxide concentrations (10-45 mM) the oxidation rate of cytochrome a increased linearly with the concentration of H2O2 (k = 700 M-1.S-1). Upon extrapolation to zero H2O2 concentration an intercept with a value of 16 s-1 (at 20 degrees C and pH 7.4) was found. A reaction sequence is described to explain these results; according to this model the rate constant (16 S-1) at zero H2O2 concentration represents the true value of the rate of electron transfer from cytochrome a to cytochrome a3 when the a3-CuB site is oxidised and unligated. However, when a complex of hydrogen peroxide with oxidised cytochrome a3 is formed, this rate is strongly enhanced. The slope (700 M-1.S-1) would then represent the rate of cytochrome a3(3+)-H2O2 complex formation. From experiments in which the pH was varied, we conclude that the reaction of H2O2 with cytochrome a3(2+) is independent of pH, whereas the electron-transfer rate from cytochrome a to cytochrome a3 gradually decreases with increasing pH. From the temperature dependence we could calculate values of 23 kJ.mol-1 and 45 kJ.mol-1 for the activation energies of the oxidations by H2O2 of cytochrome a3(2+) and cytochrome a2+, respectively. The similarity of the values that were obtained for cytochrome a oxidation both with H2O2 and with O2 as the electron acceptor suggests that the reactions share the same mechanism. In 2H2O the reactions studied decreased in rate. For the reaction of 2H2O2 with reduced cytochrome a3 in 2H2O, a small effect was found (15% decrease in rate constant). However, the internal electron-transfer rate from cytochrome a to cytochrome a3 decreased by 50%, Our results suggest that the internal electron transfer is associated with proton translocation.

Animals↗
PubMed1977

Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.

Anaerobiosis↗
PubMed1976

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.

Animals↗
PubMed1987

Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d.

Three chimeric cytochrome P450 cDNAs were constructed by replacing the central region, carboxy-terminal region, or both central and carboxy-terminal regions of cytochrome P450c cDNA with the corresponding regions of cytochrome P450d cDNA. These were inserted between the alcohol dehydrogenase I promoter and terminator of yeast expression vector pAAH5 to form expression plasmids pACDC2, pACCD1, and pACDD2. On introduction of each of these plasmids into Saccharomyces cerevisiae AH22 cells, chimeric cytochrome P450 proteins were expressed in AH22/pACDC2, AH22/pACCD1, and AH22/pACDD2 cells at the level of at least 10(5), 4 X 10(5) molecules per cell, respectively. The reduced CO-difference spectra showed that AH22/pACCD1 and AH22/pACDD2 cells contained 4 X 10(5) and 10(5) molecules per cell of the corresponding chimeric cytochrome P450 hemoproteins, designated as cytochrome P450ccd and cytochrome P450cdd, respectively. Cytochrome P450ccd exhibited higher monooxygenase activities toward 7-ethoxycoumarin, acetanilide, and benzo[alpha]pyrene than cytochrome P450c, although the substrate specificity of cytochrome P450ccd seemed to be the same as that of cytochrome P450c. Cytochrome P450cdd exhibited lower activities toward 7-ethoxycoumarin and benzo[alpha]pyrene, and a higher activity toward acetanilide as compared with those of cytochrome P450c and cytochrome P450ccd. Therefore, the substrate specificity of cytochrome P450cdd seemed to be the same as that of cytochrome P450d. These results suggest that the central one-third region of cytochrome P450c and cytochrome P450d is responsible for substrate-binding, and that the carboxy-terminal third of both cytochromes P450 plays an important role in electron transport.

Animals↗
PubMed1988

Kinetics of the interaction of cytochrome c oxidase of Paracoccus denitrificans with Paracoccus and mitochondrial cytochrome c.

We have studied the reactions of the oxidase of Paracoccus dentrificans with its membrane-bound cytochrome c and with soluble cytochrome c550 of Paracoccus and of bovine heart. The turnover rate of Paracoccus oxidase with membrane-bound cytochrome c is high, approaching 1000/sec. at 25 degrees. When soluble cytochrome c is added to the electron transport chain oxidizing NADH or succinate, no increase in 02 uptake is observed. When the oxidase is reacting with the membrane-bound cytochrome c, the reaction site is not exposed for reaction with soluble cytochrome c. We have purified the Paracoccus oxidase, following relatively simple methodology. It has three major subunits similar in molecular weight to those of the larger subunits of the bovine oxidase. When reconstituted in the presence of asolectin, it is just as active as the intact membrane-bound oxidase in reaction with soluble cytochrome c. The soluble cytochrome c reacts directly with the cytochrome aa3. We found direct evidence that the oxidase is stimulated in the presence of low concentrations of cytochrome c. The stimulatory effect could be the explanation for the so-called "high affinity" site for reaction with cytochrome c. The reaction of bovine cytochrome c with Paracoccus oxidase resembles that with the bovine oxidase in every way tested. The Paracoccus oxidase must have a cytochrome c binding site equivalent to that of the bovine enzyme. The reaction of the Paracoccus oxidase with its own soluble cytochrome c550, which has a highly negative hemisphere on the side of the molecule away from the heme crevice, has different properties from those seen in its reaction with bovine cytochrome c. However the properties all change to be like those with bovine cytochrome c on addition of poly-L-lysine. These data emphasize the importance of all of the charged groups on the cytochrome c in influencing binding or electron transfer reactions. The respiratory chain on the membranes of a cytochrome c-deficient mutant can reduce cytochrome aa3 using NADH as substrate in a manner similar to that of the wild type, although at somewhat lower rate, suggesting diffusional encounter of the large complexes within the membrane. Our data permit speculations about the possible evolution from the bacterial to the mitochondrial electron transport system.

Animals↗
PubMed1976

Multiple forms of rat liver cytochrome P-450. Immunochemical evidence with antibody against cytochrome P-448.

Purified hepatic cytochrome P-448 from 3-methylcholanthrene-treated rats was used to produce antibody in rabbits. The cytochrome P-448 antibody (IgG fraction) isolated from immune rabbit serum is quite specific and precipitates purified rat liver cytochrome P-448 at low antibody to protein ratios when assayed by the Ouchterlony double diffusion technique. Purified hepatic cytochrome P-450 from phenobarbital-treated rats cross-reacts poorly with the cytochrome P-448 antibody as do purified rabbit hepatic cytochrome P-448 and P-450. No cross-reaction is observed with purified cytochrome P-450 from beef adrenal mitochondria or from Pseudomonas putida in Ouchterlony double diffusion experiments. The cytochrome P-448 antibody produces a single distinct precipitin band with purified rat cytochrome P-448. In contrast, purified liver cytochrome P-450 from phenobarbital-treated rats gives three precipitin bands, all of which contain hemeprotein as judged by benzidine staining. At least two of the three precipitin bands are immunochemically different from the precipitin band formed with cytochrome P-448. When added to the reconstituted system, the cytochrome P-448 antibody inhibits purified rat cytochrome P-448- and P-450-supported N-demethylation of benzphetamine, O-deethylation of ethoxycoumarin, hydroxylation of benzo[a]pyrene, and the hydroxylation of testosterone at the 6beta, 7alpha, and 16alpha positions. Antibody inhibits cytochrome P-448-supported metabolism more than cytochrome P-450-supported metabolism except for benzo[a]pyrene hydroxylation at low antibody to hemeprotein ratios. In addition, the pattern and extent of inhibition of the cytochrome P-450 system depends on the substrate used, suggesting that multiple forms of the hemeprotein are present in the purified preparation from phenobarbital-treated rats. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicate that (a) cytochrome P-448 from 3-methylcholanthrene-treated rats is immunochemically different from cytochrome P-450 from phenobarbital-treated rats, and (b) there appear to be at least three hemeprotein forms in the purified cytochrome P-450 preparation from phenobarbital-treated rats.

Animals↗
PubMed1999

An engineered cytochrome b6c1 complex with a split cytochrome b is able to support photosynthetic growth of Rhodobacter capsulatus.

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.

Cytochrome b Group↗
PubMed1985

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5.

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.

Animals↗
PubMed2003

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test.

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.

Cytochrome P-450 CYP2E1↗
PubMed1989

Reduction of 7-alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their O-dealkylation by rat liver microsomal cytochrome P450.

Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy- and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of approximately 30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochrome P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b5, and could not be ascribed to an artifact of the method used to measure the formation of resourufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphatamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a3 2+ . CO.

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity. Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a3 in the mixed-valence enzyme after photodissociation. Upon addition of CO to partially reduced formate cytochrome c oxidase (a2+a3 3+ . HCOOH) the cytochrome a3 2+. CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a3 and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a3 3+ . HCOOH compound is dissociated slowly with a concomitant formation of the a3 2+ . CO compound and oxidation of cytochrome a. When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a3 and the a2+ 3 . CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.

Animals↗
PubMed1976

Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.

1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase.

Animals↗
PubMed1982

Lipid-protein interactions as determinants of activation or inhibition by cytochrome b5 of cytochrome P-450-mediated oxidations.

Activation or inhibition by cytochrome b5 of benzphetamine N-demethylation was studied in micelle-reconstituted systems containing cytochrome P-450 LM2, NADPH-cytochrome P-450 reductase, and dilauroyl-phosphatidylcholine. The effects of cytochrome b5 were critically dependent on both protein:protein and lipid:protein ratios. A 200% stimulation of N-demethylation by cytochrome b5 was obtained at cytochrome P-450 reductase:cytochrome P-450 ratios similar to those in microsomes, compared to only a 20% stimulation at a ratio of 1:1. At lipid:protein ratios less than 50:1, the addition of cytochrome b5 caused significant inhibition of benzphetamine N-demethylation. Such an inhibition could be partially reversed by increasing phospholipid content of micelles and was not seen in vesicle-reconstituted systems at cytochrome b5:cytochrome P-450 ratios of 1:1 or lower. At high cytochrome P-450 reductase:cytochrome P-450 ratios, addition of cytochrome b5 did not alter the efficiency (80%) with which NADPH was utilized: however, at ratios similar to those in microsomes, an increase in efficiency from 42% to 80% was observed. The function of cytochrome b5 was interpreted in terms of a model in which inhibition of cytochrome P-450-mediated reactions results from changes in phospholipid-protein interactions and activation occurs via facilitation of electron transfer between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane.

Animals↗
PubMed1995

Enzymic activities of covalent 1:1 complexes of cytochrome c and cytochrome c peroxidase.

We have obtained several cysteine mutants in or around the cytochrome c peroxidase binding domain of rat and yeast iso-1 cytochrome c by site-directed mutagenesis. These cysteine residues were specifically labeled with the bifunctional photoactive cross-linker 4-azidophenacyl bromide (APB). 1:1 covalent complexes of cytochrome c peroxidase and cytochrome c were generated by cross-linking these specifically labeled cytochromes c to cytochrome c peroxidase, and the 1:1 complexes were purified. Steady-state kinetic studies of the purified 1:1 complexes with free yeast and horse cytochromes c showed the following: (1) Cytochrome c peroxidase has two distinct catalytic sites--a high-affinity and a low-affinity site. (2) Other than the difference in affinity, the binding of substrate at the low-affinity site is similar to that at the high-affinity site, with yeast cytochrome c interacting more strongly than the horse protein, the binding of both substrates being sensitive to ionic strength, and both sites able to transfer electrons. (3) HPLC chromatography of purified 1:1 complex showed multiple forms of 1:1 complexes, supporting the idea of multiple possible interactions between cytochrome c and the high-affinity site on cytochrome c peroxidase. (4) An allosteric or electrostatic effect exists between the two substrate binding sites, the binding of cytochrome c to the high-affinity site decreasing the binding affinity of the low-affinity site to cytochrome c. The higher the equilibrium binding affinity of the mutant cytochrome c to the peroxidase, the larger the apparent allosteric/electrostatic effect when that mutant protein is covalently bound to the high-affinity site of the enzyme. Furthermore, different locations of the covalently bound cytochrome c at the high-affinity site on the enzyme surface result in different degrees of allosteric/electrostatic effect. The presence of two active sites on the enzyme allows a simple interpretation of some of the differences in the steady-state kinetic behavior of cytochrome c peroxidase with horse and yeast iso-1 cytochrome c.

Allosteric Regulation↗
PubMed1993

The interaction between cytochrome c2 and the cytochrome bc1 complex in the photosynthetic purple bacteria Rhodobacter capsulatus and Rhodopseudomonas viridis.

The rates of electron transfer from a ubiquinol analogue to cytochrome c2 catalyzed by the cytochrome bc1 complexes of Rhodobacter capsulatus and Rhodopseudomonas viridis were measured as a function of ionic strength. The effects of ionic strength on the kinetic parameters for the reactions are consistent with a role for electrostatic complex formation between cytochrome c2 and the cytochrome bc1 complex in the electron-transfer pathways in both photosynthetic purple non-sulfur bacteria. Additional support for a docking model in which positively charged lysines on cytochrome c2 interact with negatively charged groups on the Rb. capsulatus cytochrome bc1 complex was obtained from kinetic experiments using Rb. capsulatus cytochrome c2 and equine cytochrome c in which specific lysine residues were altered by site-directed mutagenesis and chemical modification, respectively. Equine cytochrome c, which is a poor electron donor to the reaction center of Rps. viridis, is an effective electron acceptor for the Rps. viridis cytochrome bc1 complex. Chemical modification of lysine residues on Rps. viridis cytochrome c2 has a substantially greater effect on the reduction of the Rps. viridis reaction center by ferrocytochrome c2 than on the oxidation of the Rps. viridis cytochrome bc1 complex by ferricytochrome c2. These data suggest that the docking site for Rps. viridis cytochrome c2 on the Rps. viridis reaction center tetraheme subunit differs in structure from the docking site for the cytochrome on the Rps. viridis cytochrome bc1 complex to a significant extent. In this respect, Rps. viridis differs from photosynthetic purple non-sulfur bacteria in which the reaction center does not contain a tetraheme subunit, where the binding sites for cytochrome c2 on the reaction center and the cytochrome bc1 complex appear to be quite similar.

Chromatography, Gel↗
PubMed2002

Mutational analyses of the photosynthetic reaction center-bound triheme cytochrome subunit and cytochrome c2 in the purple bacterium Rhodovulum sulfidophilum.

The purple photosynthetic bacterium Rhodovulum sulfidophilum has an unusual reaction center- (RC-) bound cytochrome subunit with only three hemes, although the subunits of other purple bacteria have four hemes. To understand the electron-transfer pathway through this subunit, three mutants of R. sulfidophilum were constructed and characterized: one lacking the RC-bound cytochrome subunit, another one lacking cytochrome c(2), and another one lacking both of these. The mutant lacking the RC-bound cytochrome subunit was grown photosynthetically with about half the growth rate of the wild type, indicating that the presence of the cytochrome subunit, while not indispensable, is still advantageous for the photosynthetic electron transfer to support its growth. The mutant lacking both the cytochrome subunit and cytochrome c(2) showed a slower rate of growth by photosynthesis (about a fourth of that of the wild type), indicating that cytochrome c(2) is the dominant electron donor to the RC mutationally devoid of the cytochrome subunit. On the other hand, the mutant lacking only the cytochrome c(2) gene grew photosynthetically as fast as the wild type, indicating that cytochrome c(2) is not the predominant donor to the RC-bound triheme cytochrome subunit. We further show that newly isolated soluble cytochrome c-549 with a redox midpoint potential of +238 mV reduced the photooxidized cytochrome subunit in vitro, suggesting that c-549 mediates the cytochrome c(2)-independent electron transfer from the bc(1) complex to the RC-bound cytochrome subunit. These results indicate that the soluble components donating electrons to the RC-bound triheme cytochrome subunit are somewhat different from those of other purple bacteria.

Alphaproteobacteria↗