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PubMed1986

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a.

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.

1-Butanol↗
PubMed1980

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex.

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa3 and to the (porphyrin) cytochrome c-cytochrome aa3 complex. 2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2.10(8) M-1.s-1 and about 10(7) M-1.S-1 (I=8.8 mM, pH 7.0, 10 degrees C), respectively. 3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa3 reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa3. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa3 has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa3 has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.

Animals↗
PubMed1982

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase.

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.

Animals↗
PubMed1977

Formation of a complex between yeast L-lactate dehydrogenase (cytochrome b2) and cytochrome c. Ultracentrifugal and gel chromatographic analyses.

Yeast L-lactate dehydrogenase formed a stable complex with cytochrome c in weakly alkaline solution of low ionic strength. The binding ratio of cytochrome c to the enzyme depended on whether free cytochrome c was present: In the presence of a micromolar concentration of cytochrome c the enzyme formed a complex with about two molecules of cytochrome c, whereas the enzyme was in a 1:1 molecular complex after removal of free cytochrome c. This suggests that the binding of one molecule of cytochrome c changes the affinity of the other binding site on the enzyme for cytochrome c. The enzyme consists of four presumably identical subunits, each containing a binding site for cytochrome c. Thus, present data confirm the concept of negative cooperativity between the subunits of the enzyme molecule in their interaction with cytochrome c.

Candida↗
PubMed1986

Immunocytochemical localization of cytochrome P-450 in hepatic and extra-hepatic tissues of the rat with a monoclonal antibody against cytochrome P-450 c.

The cellular distribution of cytochrome P-450 has been studied in the liver and a number of extrahepatic tissues in the rat by immunocytochemistry, using an antibody raised against cytochrome P-450 form c. Immunoreactive cytochrome P-450, most probably form c, was found in the proximal tubules of the kidney, in the Clara cells of the lung, and in the olfactory epithelium and Bowman's glands of the olfactory tissue, in addition to its location in the liver. Immunoreactive cytochrome P-450 was not found in the small intestine, the testes or the adrenal gland, although these organs are known to contain isoenzymes of cytochrome P-450. The use of antibody titration enabled the effects of phenobarbitone, beta-naphthoflavone and clofibrate on the content and distribution of immunoreactive cytochrome P-450 to be studied in both the liver and in the other organs discussed. Phenobarbitone induces epitope-specific cytochrome P-450 in the centrilobular cells of the liver but has no effect in any of the other tissues studied. Clofibrate is without effect on the levels of immunoreactive cytochrome P-450 in any of the tissues studied. In contrast, beta-naphthoflavone induces immunoreactive cytochrome P-450 in the periportal region of the liver, and also in the Clara cells of the lung, in the enterocytes of the small intestine and in the proximal tubules of the kidney. Of all of the tissues studied, in which immunoreactive cytochrome P-450 could be detected, only the olfactory epithelium failed to undergo enzyme induction following treatment with beta-naphthoflavone.

Adrenal Glands↗
PubMed1984

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase.

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.

Animals↗
PubMed2004

Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase.

Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways.

Animals↗
PubMed1999

Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c.

Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method. Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated. For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn). In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.

Algorithms↗
PubMed1992

Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.

Carbon Monoxide↗
PubMed1982

Identification and quantitation of a 2.0-kilobase messenger ribonucleic acid coding for 3-methylcholanthrene-induced cytochrome P-450 using cloned cytochrome P-450 complementary deoxyribonucleic acid.

We have used a plasmid containing DNA complementary to one of the two size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450 from rat liver to characterize and quantitate that mRNA. The plasmid used was constructed and identified as follows: Total poly(A+) RNA from 3-methylcholanthrene-induced liver was used as a template for cDNA synthesis. Double-stranded cDNA was inserted into plasmid pBR322 by the G-C tailing procedure. Recombinants were screened by colony hybridization using as probe [32P]cDNA prepared from partially purified cytochrome P-450 mRNA. A differential screening approach was used in which duplicate filters were hybridized with probe from either 3-methylcholanthrene treated or untreated rats. Plasmid p23 was strongly positive by colony hybridization. It was conclusively shown to contain cytochrome P-450 cDNA sequences by demonstrating that the mRNA which specifically hybridized to nitrocellulose-bound plasmid p23 could be translated in vitro into peptides that were immunoprecipitable with monoclonal antibodies specific for 3-methylcholanthrene-induced cytochrome P-450. The size and quantity of the mRNA complementary to plasmid p23 were determined by hybridization of the 32P-labeled plasmid to rat liver RNA that had been fractionated by electrophoresis under fully denaturing conditions and transferred to diazobenzyl-oxymethyl-paper. Plasmid p23 hybridized strictly to a single size of mRNA that was about 2000 nucleotides in length, the smaller of the two size classes of mRNA coding for 3-methylcholanthrene-induced cytochrome P-450. From this we concluded that, at least within the region of the mRNA contained within the insert of plasmid p23, the two size classes of 3-methylcholanthrene-induced cytochrome P-450 mRNA were very different in sequence. The mRNA complementary to plasmid p23 was increased about 4-fold after in vivo administration of 3-methylcholanthrene under conditions that result in maximal induction of 3-methylcholanthrene-induced cytochrome P-450 enzymatic activity. This increase in cytochrome P-450 mRNA parallels the increase in cytochrome P-450 enzymatic activity observed after 3-methylcholanthrene administration. These data suggest that the regulation of mRNA levels is an important point of control of cytochrome P-450 gene expression.

Animals↗
PubMed1989

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism.

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

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

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

Animals↗
PubMed1975

Conformational and functional studies of chemically modified cytochromes: N-bromosuccinimide- and formyl-cytochromes c.

N-bromosuccinimide-cytochromes c (Myer, Y. P. (1972), Biochemistry 11, 4195) and formyl-cytochrome c (Aviram, I and Schejter, A. (1971), Biochim. Biophys. Acta 229, 113) have been chromatographically purified, and the resulting components have been characterized in terms of their structure, conformation, and function. The activity measurements are considered in terms of the oxidizability, as the transference of an electron to solubilized cytochrome c oxidase, and reducibility, as the tendency to accept an electron from NADH-cytochrome c reductase. Conformational characterization has been carried out by absorption measurements, pH-spectroscopic behavior, circular dichroism, thermal denaturation, ionization of phenolic hydroxyls, the tendency to form the CO complex, and autoxidation with molecular oxygen. NBS-cytochrome c yields two major components, the relative proportions of which, with increasing modification of the protein, exhibit a pattern typical of the formation of the two in a consecutive manner. The first product contains the modification of the Trp-59 and Met-65 side chains, and the second contains the added modification of Met-80. The former in both valence states of iron is more or less like the native protein, except for an apparently slightly loosened heme crevice; the latter, as in other modifications involving modification of centrally coordinated Met-80, was found to be in a conformational state characteristic of the native protein with a disrupted central coordination complex, a loosened heme crevice, and small, but finite derangement of the polypeptide conformation. Functionally, the first component reflected 55% of the reducibility property and an unimpaired oxidizability property, while the latter exhibited derangement of both aspects of cytochrome c activity. Formyl-cytochrome c yielded a single component with modification of Trp-59. Conformationally, in both valence states, it is a molecular form with a disrupted central coordination complex, a loosened heme crevice, and gross derangement of the overall protein conformation. It exhibits a minimal reducibility property, 12%, whereas it retains a native-like tendency to transfer an electron to cytochrome c oxidase. The data from the NBS-cytochrome c components are analyzed with reference to the two forms in the earlier studies of the unpurified preparations. The results are found to be in agreement with one another. The selectivity between the reducibility and the oxidizability exhibited by the first NBS component and formyl-cytochrome c, irrespective of significant differences in the conformational and coordinational configurations of the two, has been viewed in light of a two-path, two-function model for oxidoreduction, as well as with reference to conformational and structural requirements for the oxidizability and reducibility properties of the molecule.

Binding Sites↗
PubMed1996

Stopped-flow, laser-flash photolysis studies on the reactions of CO and O2 with the cytochrome caa3 complex from Bacillus subtilis: conservation of electron transfer pathways from cytochrome c to O2.

The reaction of CO and O2 with fully reduced cytochrome caa3 from Bacillus subtilis has been studied by rapid reaction spectrophotometry. The fully reduced caa3 complex reacts with CO to give a spectrum that is characteristic of formation of ferrocytochrome a3-CO. This adduct is photosensitive, and its recombination rate is proportional to CO concentration with a bimolecular value of 1.2 x 10(5)M-1 s-1. When the CO compound of the reduced complex is exposed to O2, the rate of oxidation proceeds at 0.1 s-1, which is assigned as the CO off rate. These kinetic constants give an equilibrium dissociation constant for the CO complex of 0.83 microM. Photolysis of the CO adduct in the presence of O2 reveals three reaction phases over the first 3 ms and an additional phase on the second time scale. A kinetic model is proposed in which fully reduced oxidase first combines with O2 and then electron transfer commences from both cytochrome a and a3, followed rapidly by electron input from CuA and the cytochrome c domain. An equivalent kinetic model has been used to account for the reactivity of mammalian cytochrome c oxidase in its electrostatic complex with soluble cytochrome c [Hill, B. C., (1994) J. Biol. Chem. 269, 2419-2425]. However, unlike the mitochondrial complex, the reactivity of cytochrome c in the B. subtilis caa3 complex is unaffected by ionic strength. Thus the cytochrome c moiety in the B. subtilis caa3 complex seems to be fixed in a reactive orientation by its covalent association with the rest of the oxidase complex. The pathway of electron transfer from cytochrome c to O2 appears very well conserved from B. subtilis to the mammalian respiratory chain, making the B. subtilis protein a good model to probe intersite electron transfer within the cytochrome c-cytochrome oxidase complex.

Bacillus subtilis↗
PubMed2007

Serum cytochrome c detection using a cytochrome c oxidase biosensor.

Within the last ten years it has emerged that the release of cytochrome c plays a critical role in the important process of programmed cell death. It has also been shown that this protein is released into the circulating blood following MIs (myocardial infarctions). Methods for the detection of this protein have therefore become important. The enzyme cytochrome c oxidase is specific for cytochrome c. Bovine cytochrome c oxidase was successfully immobilized in a didodecyldimethylammonium bromide vesicular system on to gold electrodes and its interaction with cytochrome c was studied. Square-wave-voltammetric analysis of the biosensor showed two redox couples with midpoint potential, E(0)', values of +182 and +414 mV compared with Ag/AgCl. The redox couple with E(0)' of +414 mV showed a cathodic sensitivity to the presence of cytochrome c in both buffer solution and human serum. Responses of the cytochrome c oxidase biosensor to oxidized cytochrome c followed hyperbolic electrochemical Michaelis-Menten kinetics with a K(m) of 1.57 microM and maximum current (I(max)) of 1.38 x 10(-6) muA. The detection limit of the biosensor in human serum was 0.2 microM, which is well below the lowest physiological concentration of 0.8 muM previously reported for MIs [Alleyne, Joseph and Sampson (2001) Appl. Biochem. Biotechnol. 90, 97-105]. These results indicate that the cytochrome c oxidase biosensors could be used to determine variations in cytochrome c concentration and thus have potential to be used as a diagnostic tool in the detection of MIs and possibly also in the study of programmed cell death.

Biosensing Techniques↗
PubMed1986

NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions.

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1994

Electron transfer from Phanerochaete chrysosporium cellobiose oxidase to equine cytochrome c and Pseudomonas aeruginosa cytochrome c-551.

The electron-transfer reactions of cellobiose oxidase (CBO) have been investigated by conventional and by rapid-scan stopped-flow spectroscopy at pH 6.0. Analysis of the absorbance/time/wavelength matrix by Singular Value Decomposition (SVD) confirms earlier studies showing that cellobiose rapidly reduces the flavin group (7.7 s-1; cellobiose, 100 microM) which in turn slowly (0.2 s-1) reduces the cytochrome b moiety. In the presence of CBO, cellobiose reduces cytochromes c in a reaction that does not depend on oxygen or superoxide. The rate limit for this process is independent of the source of the cytochromes c and is identical with the rate of cytochrome b reduction. Rapid-mixing experiments show that cytochrome b may donate electrons very rapidly to either mammalian cytochrome c or bacterial cytochrome c-551. The reactions were second-order (kc = 1.75 x 10(7) M-1 x s-1; kc-551 = 4.3 x 10(6) M-1 x s-1; pH 6.0, 21 degrees C and I0.064) and strongly ionic-strength (I)-dependent: kc decreasing with I and kc-551 increasing with I. These results suggest the electron-transfer site near cytochrome b bears a significant negative charge. Equilibrium gel chromatography confirms that CBO oxidase and positively charged mammalian cytochrome c make stable complexes. These results are discussed in terms of a model suggesting an electron-transfer role for cytochrome b in vivo, possibly connected with radical-mediated cellulose breakdown.

Animals↗
PubMed1995

Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c.

Subunit V, one of the nuclear-coded subunits of yeast cytochrome c oxidase, has two isoforms, Va and Vb. These alter the in vivo intramolecular rates of electron transfer within the holoenzyme (Waterland, R. A., Basu, A., Chance, B., and Poyton, R. O. (1991) J. Biol. Chem. 266, 4180-4186). The isozyme with Vb has a higher turnover rate and a higher intramolecular transfer rate than the isozyme with Va. To determine how these isoforms affect catalysis, we have examined their effects on the binuclear reaction center and on the interaction between cytochrome c oxidase and the two isoforms, iso-1 and iso-2, of yeast cytochrome c. Infrared spectroscopy of carbon monoxide liganded to heme a3 has revealed a single conformer for the binuclear reaction center in the isozyme with Vb but two discrete conformers in the isozyme with Va. The kinetics of interaction for all four pairwise combinations of isozymes with each subunit V isoform and the two cytochrome c isoforms are biphasic, with high and low affinity electron transfer reactions. In general, the isoforms of cytochrome c and subunit V do not alter the Km but do affect the TNmax. The TNmax for isozymes carrying Vb are higher at both high and low affinity sites for each cytochrome c isoform. Iso-1-cytochrome c supports a higher TNmax than Iso-2-cytochrome c. Surprisingly, the combinatorial effect of both sets of isoforms on TNmax is minimized with the pairs of isoforms (iso-1-cytochrome c and subunit Va or iso-2 and subunit Vb) that are co-expressed in cells. Together, these findings support the conclusion that the subunit V isoforms modulate catalysis and suggest that they do so by affecting the environment or structure of the binuclear reaction center. They also suggest that the coexpression of the two cytochrome c isoforms with two subunit V isoforms serves to minimize differences in electron transfer rates brought about by the subunit V isoforms.

Cytochrome c Group↗