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The cytochromes P450 are a large class of heme-containing enzymes that catalyze a broad range of chemical reactions in biosystems, mainly through oxygen-atom transfer to substrates. A relatively unknown reaction catalyzed by the P450s, but very important for human health, is the activation of halogenated substrates, which may lead to toxicity problems. However, its catalytic mechanism is currently unknown and, therefore, we performed a detailed computational study. To gain insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and reductive P450-mediated activation of tetra- and trichloromethane as halogenated models with density functional theory (DFT) methods. We propose an oxidative halosylation mechanism for CCl4 under aerobic conditions by Compound I of P450, which follows the typical Groves-type rebound mechanism. By contrast, the metabolism of CHCl3 occurs preferentially via an initial hydrogen-atom abstraction rather than halosylation. Kinetic isotope effect studies should, therefore, be able to distinguish the mechanistic pathways of CCl4 versus CHCl3. We find a novel mechanism that is different from the well accepted P450 substrate activation mechanisms reported previously. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized using Marcus theory equations, which reproduce experimental product distributions. [ABSTRACT FROM AUTHOR]

Summary Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification. [ABSTRACT FROM AUTHOR]

Background and Purpose: Certain nuclear receptors (NRs) such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR) mediate induction of some cytochrome P450 enzymes and ABC transporters but conflicting reports exist. The purpose of this study was to assess the reasons for these discrepancies and use a standardized approach to compare activators of NRs.Experimental Approach: Dexamethasone, pregnenolone 16alpha-carbonitrile, rifampicin, phenobarbital and chenodeoxycholic acid were incubated with HepG2, Caco-2 and cryopreserved human hepatocytes prior to analysis of mRNA and protein for CYP2B6, CYP3A4, CYP3A5, ABCB1, ABCC1, ABCC2, PXR, CAR and FXR.Key Results: Dexamethasone significantly up-regulated PXR, CYP3A4 and ABCB1 expression in HepG2 and Caco-2 cells. As a result, including dexamethasone as a media supplement masked the induction of these genes by pregnenolone 16alpha-carbonitrile, which may explain discrepancies between previous reports. In the absence of dexamethasone, significant activator-dependent induction was observed in all cell types. Significant correlations were observed between the fold increase in mRNA and in protein, which were, for most instances, logarithmic. Changes in mRNA expression were greater in cell lines than primary cells but for most transcripts correlations were observed between fold increases in HepG2 and hepatocytes.Conclusions and Implications: Clearly, no in vitro system can imitate the physiology of a hepatocyte or intestinal cell within an intact organ in vivo, but these data explain some of the discrepancies reported between laboratories and have important implications for study design. [ABSTRACT FROM AUTHOR]

Cytochrome P450 proteins are involved in metabolism of drugs and xenobiotics. In the endoplasmic reticulum a single nicotinamide adenine dinucleotide phosphate (NADPH) P450 oxidoreductase (POR) supplies electrons to all microsomal P450s for catalytic activity. POR is a flavoprotein that contains both flavin mononucleotide and flavin adenine dinucleotide as cofactors and uses NADPH as the source of electrons. We have recently reported a number of POR mutations in the patients with disordered steroidogenesis. In the first report we had described missense mutations (A287P, R457H, V492E, C569Y, and V608F) identified in four patients with defects in steroid production. Each POR variant was produced as recombinant N-27 form of the enzyme in bacteria and as full-length form in yeast. Membranes from bacteria or yeast expressing normal or variant POR were purified and their activities were characterized in cytochrome c and CYP17A1 assays. Later we have published a larger study that described a whole range of POR mutations and characterized the mutants/polymorphisms A115V, T142A, M263V, Y459H, A503V, G539R, L565P, R616X, V631I, and F646del from the sequencing of patient DNA. We also studied POR variants Y181D, P228L, R316W, G413S, and G504R that were available in public databases or published literature. Three-dimensional structure of rat POR is known and we have used this structure to deduce the structure–function correlation of POR mutations in human. The missense mutations found in patients with disordered steroidogenesis are generally in the co-factor binding and functionally important domains of POR and the apparent polymorphisms are found in regions with lesser structural importance. A variation in POR can alter the activity of all microsomal P450s, and therefore, can affect the metabolism of drugs and xenobiotics even when the P450s involved are otherwise normal. It is important to study the genetic and biochemical basis of POR variants in human population to gain information about possible differences in P450 mediated reactions among the individuals carrying a variant or polymorphic form of POR that could impact their metabolism. [ABSTRACT FROM AUTHOR]

Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among individuals and, therefore, so does drug efficacy as well as susceptibility to side effects and toxicity. Thus, assessing P450 activity is of great interest in drug development and clinical pharmacology. This study investigates the phenotyping of a single P450 activity by analyzing urine samples using isocratic reverse-phase HPLC. Specifically, the activity of human P450 1A2, which converts caffeine into paraxanthine, can be investigated by measuring the change in caffeine and paraxanthine concentrations in urine over time following a single dose of caffeine. There is an observable relationship between caffeine intake and paraxanthine formation that varies among individuals. This laboratory exercise provides a means for simple assessment of P450 1A2 metabolic activity using an HPLC method without additional extraction or purification steps and introduces students to the complexities of individualized medicine as well as the basic biochemical techniques of sample preparation and quantitative HPLC. Furthermore, students may design and test their own hypothesis using these methods. [ABSTRACT FROM AUTHOR]

Cytochromes b561 are a family of transmembrane proteins found in most eukaryotic cells. Three evolutionarily closely related mammalian cytochromes b561 (chromaffin granule cytochrome b, duodenal cytochrome b, and lysosomal cytochrome b) were expressed in a Saccharomyces cerevisiaeΔ fre1Δ fre2 mutant, which lacks almost all of its plasma membrane ferrireductase activity, to study their ability to reduce ferric iron (Fe3+). The expression of each of these cytochromes b561 was able to rescue the growth defect of the Δ fre1Δ fre2 mutant cells in iron-deficient conditions, suggesting their involvement in iron metabolism. Plasma membrane ferrireductase activities were measured using intact yeast cells. Each cytochrome b561 showed significant FeCN and Fe3+-EDTA reductase activities that were dependent on the presence of intracellular ascorbate. Site-directed mutagenesis of lysosomal cytochrome b was conducted to identify amino acids that are indispensable for its activity. Among more than 20 conserved or partially conserved amino acids that were investigated, mutations of four His residues (H47, H83, H117 and H156), one Tyr (Y66) and one Arg (R67) completely abrogated the FeCN reductase activity, whereas mutations of Arg (R149), Phe (F44), Ser (S115), Trp (W119), Glu (E196), and Gln (Q131) affected the ferrireductase activity to some degree. These mutations may affect the heme coordination, ascorbate binding, and/or ferric substrate binding. Possible roles of these residues in lysosomal cytochrome b are discussed. This study demonstrates the ascorbate-dependent transmembrane ferrireductase activities of members of the mammalian cytochrome b561 family of proteins. [ABSTRACT FROM AUTHOR]

Our objective was to identify the cytochrome P450 (CYP) enzymes that metabolise pioglitazone and to examine the effects of the CYP2C8 inhibitors montelukast, zafirlukast, trimethoprim and gemfibrozil on pioglitazone metabolism in vitro. The effect of different CYP isoform inhibitors on the elimination of a clinically relevant concentration of pioglitazone (1 μM) and the formation of the main primary metabolite M-IV were studied using pooled human liver microsomes. The metabolism of pioglitazone by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5 was investigated using human recombinant CYP isoforms. In particular, the inhibitors of CYP2C8, but also those of CYP3A4, markedly inhibited the elimination of pioglitazone and the formation of M-IV by HLM. Inhibitors selective to other CYP isoforms had a minor effect only. Of the recombinant isoforms, CYP2C8 (20 pmol/ml) metabolised pioglitazone markedly (56% in 60 min.), and also CYP3A4 had a significant effect (37% in 60 min.). Montelukast, zafirlukast, trimethoprim and gemfibrozil inhibited pioglitazone elimination in HLM with IC50 values of 0.51 μM, 1.0 μM, 99 μM and 98 μM, respectively, and the formation of the metabolite M-IV with IC50 values of 0.18 μM, 0.78 μM, 71 μM and 59 μM, respectively. In conclusion, pioglitazone is metabolised mainly by CYP2C8 and to a lesser extent by CYP3A4 in vitro. CYP2C9 is not significantly involved in the elimination of pioglitazone. The effect of different CYP2C8 inhibitors on pioglitazone pharmacokinetics needs to be evaluated also in vivo because, irrespective of their in vitro CYP2C8 inhibitory potency, their pharmacokinetic properties may affect the extent of interaction. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) enzymes include a family of related enzymes that are involved in metabolism of vitamins, steroids, fatty acids, and other chemicals. This review presents a brief historical overview of the discovery and characterization of P450 enzymes extending from intermediary metabolism to the fields of drug metabolism and toxicology. Introductions to P450 enzyme structure and function are also presented. The goals of this review are to 1) provide an introduction to a few of the many aspects of P450 research relating to humans, 2) introduce additional ways of thinking about metabolism, 3) provide some basic examples of P450 enzymology, and 4) provide applications to topics widely taught in undergraduate courses in biochemistry. [ABSTRACT FROM AUTHOR]

Aim: The incidence of coronary heart diseases (CHD), congestive heart failure (CHF) and myocardial infarction is higher in diabetic patients than in non-diabetic groups, with these incidences being more in women than in the men. Hence, we examined involvement of mitochondrial energy transduction functions. Methods: Mitochondrial energy metabolism in cardiomyopathy was studied using streptozotocin (STZ)-diabetic male and female rats as the model system. Effects of insulin treatment were also evaluated. Results: The body and heart weights decreased in both male and female diabetic rats. Insulin treatments resulted in significant increase in the body and heart weights in the female rats. Mitochondrial respiration rates with all the substrates tested decreased in diabetic condition in both males and females. Treatment with two dose -regimens of insulin had differential restorative effect on mitochondrial substrate oxidation in the males but caused hyper-stimulation in the females. Diabetic state brought about 19% decrease in the cytochrome aa3 content in the female rats. Treatment with 0.6 units of insulin significantly increased the cytochrome contents in general in both the sexes whereas higher dose (1.0 unit) caused decrease in the cytochromes content in the females. Diabetic state resulted in decreased dehydrogenases activities; insulin treatments had differential effect on the dehydrogenase activity in the males and the females. The results suggest that insulin treatment-induced hyper-stimulation of respiration in female rats may lead to increased production of reactive oxygen species. Besides, increased formation of advanced glycosylated end products may further lead to increased risk of CHF and CHD. Conclusions: The results suggest that differential effects of STZ-diabetes and insulin treatments in the female rats than in males may be the underlying cause for increased incidence of diabetic cardiomypathies in the females. [ABSTRACT FROM AUTHOR]

Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membrane-bound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles. [ABSTRACT FROM AUTHOR]

In the current study, the bioconcentration behavior and acute toxicity of two polycyclic musks, Tonalide® 7-acetyl- 1,1,3,4,4,6,-hexamethyl-1,2,3,4,-tetrahydronaphthalene (AHTN) and Galaxolide® 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexa-methylcyclopenta[γ]-2-benzopyran (HHCB), were studied in two benthic organisms. Polycyclic musks are frequently used fragrances, and they have been detected in different compartments of the environment. The aim of this study was to fill some empirical data gaps for AHTN and HHCB for benthic organisms. Results show that differences exist between both organisms. Chironomus riparius exhibited bioconcentration factors (BCFs) for AHTN and HHCB substantially lower than predicted for nontransformed organics. The BCFs for both chemicals increased after coexposure of the organism to the cytochrome P450 inhibitor piperonyl butoxide. Thus, the low BCF values were the result of rapid biotransformation of AHTN and HHCB in the midge larvae. Bioconcentration kinetics indicated that both chemicals induced their own cytochrome P450-mediated metabolism. Acute toxicity of AHTN to midge larvae was reduced compared to predicted baseline toxicity and was similar for HHCB. Bioconcentration of AHTN and HHCB in the worm (Lumbriculus variegatus) is in agreement with predictions based on the octanol-water partition coefficients of these chemicals. Acute toxicity was found to be similar to predicted values for baseline toxicity. Summarizing, for AHTN and HHCB, acute toxicity and bioconcentration behavior in L. variegatus was in accordance with predicted data for nontransformed organics. In C. riparius, bioconcentration as well as toxicity were reduced. [ABSTRACT FROM AUTHOR]

We studied the estrogenic effects of model chemicals in one-year-old juvenile rainbow trout. Methoxychlor (20 mg/kg), diethylstilbestrol (15 mg/kg), 4-tert-octylphenol (25 and 50 mg/kg), and biochanin A (25 and 50 mg/kg) were injected intraperitoneally on days 1, 4, and 7. Fish were sacrificed on day 9 and examined for multiple biomarkers. All of the test chemicals caused increases in plasma vitellogenin levels, a biomarker of estrogenicity. Treatment with the xenoestrogens decreased hepatic lauric acid hydroxylase activity and, as shown by Western blots, also generally reduced expression of hepatic cytochrome P450s 2K1 (CYP2K1), 2M1 (CYP2M1), and 3A27 (CYP3A27) at the protein level. Both doses of biochanin A also significantly induced P4501A (CYP1A) and greatly increased hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity. These findings suggest that methoxychlor, diethylstilbestrol, 4-tert-octylphenol, and biochanin A were all estrogenic and mimicked 17β-estradiol (E2) in repressing the expression of cytochrome P450 isoforms (CYP2K1, CYP2M1, and CYP3A27) in the rainbow trout liver. Additionally, biochanin A was found to induce CYP1A in this fish species. [ABSTRACT FROM AUTHOR]

The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H2:heterodisulfide oxidoreductase) of Methanosarcina mazei Gö1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 values of 20 nmol DPI·mg–1 protein and 45 nmol DPI·mg–1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2‐hydroxyphenazine‐dependent reactions as catalyzed by F420‐nonreducing hydrogenase, F420H2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H2‐dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane‐bound cytochromes indicated that DPI inhibited CoB‐S‐S‐CoM‐dependent oxidation of reduced cytochromes and H2‐dependent cytochrome reduction. Membrane‐bound and purified F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2‐hydroxyphenazine were used as electron acceptors. Detailed examination of 2‐hydroxy‐phenazine‐dependent F420H2‐oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the Km value for 2‐hydroxyphenazine, which increased from 35 µm to 100 µm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron... [ABSTRACT FROM AUTHOR]

The spectral and electrochemical parameters, as well as the orientations of ibc heme planes with respect to the membrane plane, of the c-type hemes present in membrane fragments from Heliobacillus mobilis were characterised by optical and EPR spectroscopy. Cytochrome c553 was thereby shown to represent at least four and possibly five heine species with the following characteristics: Em = -60 mV ± mM, g... = 2.92, 60°= Em +90 mV±10 mM,g...=2.92, 90°; Em = +12() mV±20 mV. g... = 3.02; and Em = +170 mV ±2o mM, g... = 3.03. The latter component may correspond to two hemes with redox midpoint potentials of Em= +160 mV ±20 mV and mV=+180 mV ±20 mV (all Em, values at pH 7.0). For the heme species having g... peaks at g≈3.03. determination of individual orientations was precluded due to the superposition of several differently oriented hemes. About one copy of each heine was found to be present per photosynthetic reaction centre, with the exception of the +120 mV componero for which a stoichiometry of 2 hemes/reaction centre was oblained. The heine proteins were detergent-solubilised and partially purified. Three c-type cytochromes that migrated with apparent molecular masses of 18. 29 and 50 kDa were detected on SDS/PAGE. Optical redox titrations at pH 7.0 showed redox midpoint potentials of + 160 mV ± 10 mV for the 18-kDa cytochrome, and - 60 mV 2± l0 mV. with possible contributions around +160 mV for the 50-kDa cytochrome. A tentative attribution of heine species observed in membranes to the isolated heine proteins is presented. The results obtained on H. mobilis are compared with those reported for green sulphur bacteria. [ABSTRACT FROM AUTHOR]

In many tissues from different species, pregnenolone and dehydroepiandrosterone (DHEA) are hydroxylated mainly at the 7α position by a cytochrome P450 (P450)-containing microsomal enzyme complex. In addition. 7-hydroxysteroids have been shown to activate immune processes in mice. The reported production of 7β-hydroxypregnenolone and 7β-hydroxy-DHEA was not Supported by formal identification, and the P450 responsible for 7α-hydroxylation and 7β-hydroxylation of pregnenolone and DttEA have not been identified. Based on results of analyses by cryslallizatin to constant specific activity and gas chromatography/mass spectrometry, we report that mouse-liver and mouse-brain microsomes carried out 7β-hydroxylation of pregnenolone and DHEA, and that yeast-expressed mouse cytochrome P4501A1 (P450 1A) transformed pregnenolone into 7β-hydroxypregnenolone [Km = 25.1 ± 0.4 µM, turnover number = 979±30 pmol · min-1· nmol-1 mouse P450 1A1 ). Neither 7-hydroxy derivatives of DHEA nor 7α-hydroxypregnenolone was produced by P450 1A1. The presence of P450 IAI m liver and brain microsomes was shown by Western blot analysis, and induction of mouse P450 1Al by β-naphthoflavone resulted in increased 7β-hydroxylation of pregnenolobe in liver microsomes. Studies of the brain-microsome 7β-hydroxylating enzyme with pregnenolone or DHEA gave Km of 5.0 µM and 4.9 µM, respectively, and Vmax of 4.5 pmol · min-1 · mg-1 and 6.1 pmol · min-1 · mg-1, respectively, and showed the absence of cross-inhibitions between the two steroids. These findings indicate that, in addition to unidentified P450, P450 IAI is involved in 7β-hydroxylation of pregnenolone and may contribute in part to the production of the 7-hydroxylated steroids necessary for activation of immune defenses. [ABSTRACT FROM AUTHOR]

A novel strategy which employs chromatography on an immobilized metal ion has been developed for the purification of bacterial cytochrome c and quinol oxidases. Many bacterial oxidase complexes appear to have a natural affinity to bind to the chelated copper ion. A combination of three different chromatographic principles (anion exchange, metal-affinity and gel filtration) makes an effective tool chest for the preparation of homogeneous and protein-chemically pure bacterial oxidases. These preparations have been used for two-dimensional crystallization. Until, now crystals have been obtained using the Paracoccus denitrificans Rhodobacter sphaeroides cytochrome aa3 and the Escherichia coli cytochrome bo. The crystals diffract to approximately 2.5 nm in negative stain and have potential for further structural studies. [ABSTRACT FROM AUTHOR]

Photosystem I and II core complexes were resolved in a single step from the thylakoid membrane of Synechocystis sp. PCC 6803 by using a mild solubilization procedure in dodecyl β-D-maltoside and Deriphat/PAGE. For each photosystem, two green bands were obtained containing oligomeric and monomeric forms of the core complexes of either photosystem. The oligomers are likely to be trimers in the case of photosystem I and dimers for photosystem II. The absorption spectra, polypeptide and pigment composition of green bands corresponding to either photosystem I or photosystem II were identical for monomeric and oligomeric forms. The cytochrome b-559 content of photosystem II was evaluated to be one cytochrome b-559/reaction center both in the monomeric and dimeric forms. Two new 15-kDa and 22-kDa carotenoid-binding proteins were isolated and their polypeptides purified to homogeneity. [ABSTRACT FROM AUTHOR]

Cytochrome cd1 nitrite reductase was isolated from magnetite-containing cells of the magnetotactic bacterium Magnetospirillum (formerly Aquaspirillum) magnetotacticum, which was microaerobically cultivated under denitrifying conditions. The enzyme showed absorption maxima at 643 nm and 409 nm in the oxidized form, and at 663, 551, 522, and 418 nm in the reduced form. A distinctive split absorption band did not occur at about 550 nm. The pyridine ferrohemochrome spectra suggested the presence of heme c and heme d1 in the molecule. The enzyme was composed of two identical subunits each with a molecular mass of 54 kDa; each subunit contained one c-type and one d-type heme. The isoelectric point was 9.2. The redox potentials of heme c and heme d1 were estimated to be +191 mV and +180 mV, respectively. Although the enzyme showed cyanide-sensitive N,N,N′,N′-tetramethyl-p-phenylenediamine-O2 oxidoreductase activity and N,N,N′,N′-tetramethyl-p-phenylenediamine-nitrite oxidoreductase activity, the enzyme did not oxidize M. magnetotacticum ferrocytochrome c-550 and Pseudomonas aeruginosa ferrocytochrome c-551 in the presence of nitrite. Furthermore, sodium succinate did not cause the reduction of cytochrome cd1 in the crude cell-flee extract prepared from the magnetite-containing bacterial cells. However, M. magnetotacticum cytochrome cd1 showed a novel Fe(II):nitrite oxidoreductase activity whereas P. aeruginosa cytochromes cd1 had no Fe(II):nitrite oxidoreductase activity. These results suggest that M. magnetotacticum cytochrome cd1 may function as a Fe(II)-oxidizing enzyme under microaerobic conditions using nitrite as electron acceptor. [ABSTRACT FROM AUTHOR]

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified. Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related m aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 μg ml-1. A similar the was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72±0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 ± 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 ± 42 electrons · s-1. Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities. [ABSTRACT FROM AUTHOR]

Studies the regulation of synthesis of cytochrome d in Escherichia coli using mutants with cytochrome-d-beta-galactosidase gene fusions. Effects of various protonophorous uncouplers when added to the growth medium; Cytochrome-d-inducing activity of incouplers; Role of cytochrome d in the sodium ions energetics.

Studies the inhibition of bovine cytochrome P-450 by 18-unsaturated progesterone derivatives. Aldosterone biosynthesis in the bovine system; Inhibition of hydroxylase activity by corticosterone; Binding of substrates and inhibitors to the active site.

Studies the cyclic voltammetry and nuclear magnetic resonance (NMR) of Rhodopseudomonas palustris cytochrome pH-dependent conformational states. Electrochemical and spectroscopic measurements; Sensitivity of the electronic spectra and the hyperfine-shifted NMR resonances to a number of acid/base equilibria; Low-pH oxidation of the protein by dioxygen.

Unlike mitochondria, many bacteria use a large repertoire of c-type cytochromes in different branches of their electron transport system. Among the many cytochromes c present in the soil bacterium Paracoccus denitrificans, a membrane-bound cytochrome (c552> has been suggested to mediate the electron transport between the cytochrome bc complex and cytochrome-c oxidase [Berry, E. A. & Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467]. We have purified this cytochrome from cytoplasmic membranes, and cloned and sequenced its gene, cycM. Sequence analysis reveals that, while its C-terminal portion is highly similar to type-I cytochromes c, its N-terminal part contains a hydrophobic segment providing membrane attachment. In addition, we present immunological evidence for its functional role in respiration. [ABSTRACT FROM AUTHOR]

Of the aromatic ¹H-NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through-space ring-current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P-450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation. As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P-45011β (CYP11B1)-dependent and P-450sec (CYP11A1)-dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin. The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary-structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent-exposed region of the molecule. [ABSTRACT FROM AUTHOR]

Analyzes the cytochrome c reductase from potato. Description of the identity of the cytochrome by immunoblotting and direct sequence determination; Immunoprecipitation of the isolated cytochrome c reductase.

Heme proton resonances have been assigned for ferricytochromes c-551 isolated from four distinct species of bacteria. While the available structure information indicates that the four cytochromes have very similar conformations in solution, including the chirality of the methionine ligand sulfur bond, the chemical shifts of the paramagnetically shifted resonances are surprisingly different, more so than has been previously reported for a homologous series of ferricytochromes. The resonances are contrasted in terms of chemical shift and the temperature dependence of the shift, which gives rise to a very strong anti-Curie effect for some specific protons. Non-methyl heme resonances do display an approximately conserved set of chemical shifts, but the heme methyl groups demonstrate a wide range of values. The 12¹ heme methyl group is always the highest frequency heme methyl, but the relative positions of the other methyl groups may change. The 7¹ heme methyl group always displayed strong anti-Curie behavior, while the 12¹ methyl group displayed normal Curie behavior. The behavior of the other methyl groups was variable. Possible reasons for the range of observations will be discussed. In spite of their NMR differences, all the ferricytochromes c-551 demonstrated comparable electron-transfer rates to a membrane-bound cytochrome reductase system. [ABSTRACT FROM AUTHOR]

Helerodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (>700 kDa) which was dissociated by dodecyl β-D-maltoside. Alter chromatography on Q-Sepharose. an active heterodisulfide reduciasc preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa, For each 69 kDa, the enzyme contained 0.6 mol cytochrome b. 0.2 mol FAD, 20 mol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating flint the cytochrome b served as electron donor for heterodisulfide reduction. [ABSTRACT FROM AUTHOR]

An electrochemical study of the periplasmic cytochrome c553 of Desulfovibrio vulgaris (Hildenborough) is presented. The dependence of the midpoint potential on temperature and pH was studied with cyclic voltammetry. The voltammograms obtained were reversible and revealed that this cytochime showed fast electron transfer on a bare glassy carbon electrode. The midpoint potential at pH 7.0 and 25°C was found to be 62 mV versus the normal hydrogen electrode. It was observed that the temperature dependence was discontinuous with a transition temperature at 32°C. The standard reaction entropy at the growth temperature of the organism (37°C) was calculated to be ΔS°'= -234 J mol-1 K-1. The pH dependence of the midpoint potential could be described with one pK of the oxidized form with a value of 10.6. The second-order rate constant for electron transfer between cytochrome c553 and the Fe-hydrogenase from D. vulgaris (H) was also determined with cyclic voltammetry. The equivalent rate constant for cytochrome c3 and hydrogenase was measured for comparison. The second-order rate constants are 2X107 M-1 s-1 for cytochrome c553 and 2x108 M-1 s-1 for cytochmme c3. The kinetic parametem of the hydrogenase for both tyrochromes were determined using the spectropholometric hydrogen consumption assay. With cytochrome c553 this resulted in a Km of 46 µM and a maximum turnover number of 7.1 X 10² s-1 in the H2 consumption assay. The values with cytochrome c3 were 17 µM and 6.4x10²s-1 respectively. The importance of the different kinetic parameters for contrasting models proposed to describe the function of the Fe-hydrogenase are discussed. [ABSTRACT FROM AUTHOR]

Investigates the side-chain specificities of human and bovine cytochromes P-450[sub scc]. Kinetics of the conversion of structural analogues of cholesterol with modified side chains to pregnenolone; Conservation of the active site of cytochrome P-450[sub scc] between bovine and human cytochromes; Species differences.

Two-dimensional NMR techniques have been used to assign proton resonances in the haem cavity of Methylophilus methylotrophus cytochrome c", a monohaem protein with bis-histidinyl ligation which has been shown to coupe electron and proton transfer. All the assignments were made directly for the oxidized paramagnetic form of the cytochrome. Nearly all of the haem protons (90%) and the protons of both axial ligands have been assigned; the side-chain protons from four other residues in the haem pocket have also been identified. The data indicate a highly symmetric unpaired-electron distribution in the haem group, which agrees with a perpendicular orientation of the axial imidazole planes. The two haem propionate groups have contrasting degrees of exposure to the solvent, with the propionate group at position 13 being highly exposed. To obtain information on the dynamics of the haem environment, measurements of the 1H/2H-exchage rates of amide protons located in the haem cavity were performed.The two faces of the haem are found to differ markedly with respect to water accessibility. All of this information, together with additional protein sequencing data, indicates that His52 remains attached upon reduction and the redox-linked protonation occurs via a channel running through the haem cleft on the opposite face. [ABSTRACT FROM AUTHOR]

Investigates the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1. Role of the cytochrome P-450 enzyme system in the monooxygenation of a broad range of endogenous substrates and xenobiotics.

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure [Salgueiro, C. A., Turner, D. L., Santos, H., LeGall, J. and Xavier, A. V. (1992) FEBS Lett., in the press]. [ABSTRACT FROM AUTHOR]

The present study was undertaken to examine the nature of the inhibitory action of glycerol on the liver microsomal monooxygenase system. In agreement with earlier observations, glycerol inhibited benzphetamine N-demethylation by liver microsomes of the phenobarbital-treated rabbit. The presence of glycerol in the medium did not affect binding of the substrate to cytochrome P450. Another polyol, ethylene glycol, was equally efficient in inhibiting benzphetamine N-demethylation. Both also inhibited reduction of rabbit cytochrome P450 LM2, cytochrome c and potassium ferricyanide by NADPH-cytochrome-P450 reductase in microsomes. Recently, we showed that the stimulation of electron transfer by increased ionic strength is due to neutralization of electrostatic interaction between NADPH-cytochrome-P450 reductase and its charged redox partners [Voznesensky, A. I. & Schenkman, J. B. (1992) J. Biol. Chem. 267, 14669–14676]. Polyols have an opposite effect to that of salt on ionic properties of a solution. They decrease the dielectric constant, thereby promoting electrostatic interactions between proteins. Addition of polyols decreased the conductivity of the medium. When rates of electron transfer to charged acceptors, cytochrome P450, cytochrome c and potassium ferricyanide, at various salt and polyol concentrations, relative to activities in 200 mM sodium phosphate, were plotted as a function of the conductivity the data for each acceptor fit on the same line. In contrast, neither alteration of ionic strength nor potyol addition affected the rate of electron transfer from NADPH-cytochrome-P450 reductase to an uncharged acceptor 1,4-benzoquinone. The data obtained is consistent with our earlier suggestion that charge repulsion limits redox interactions between rabbit cytochrome P450 LM2 and its reductase at low ionic strength, and suggest that the observed action of polyols is the result of enhancement of electrostatic interactions that inhibits electron transfer between NADPH-cytochrome-P450 reductase and its charged redox partners. In congruence with the hypothesis, the Km of rabbit cytochrome P450 LM2 for NADPH-cytochrome-P450 reductase was increased almost one order of magnitude by elevating the glycerol content from 5% to 25% (by vol.) without a change in Vmax. [ABSTRACT FROM AUTHOR]

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Schäfer, G. (1990) Eur. J. Biochem. 191, 297–305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 ↑± 10% of the enzyme concentration, while the high-spin signal accounts for only 40 ± 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 ± 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CuB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 ± 10 mV and 370 ± 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CuB(I) center, which in the CuB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 ± 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases. [ABSTRACT FROM AUTHOR]

The multicopper proteins, nitrous-oxide reductase (N2OR) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4–35 GHz. Our results support a Cu-Cu interaction in COX and N2OR. At least 10 lines in the 2.7-GHz, 12 lines in the 4.6-GHz and 14 lines in the 9.2 GHz spectra were resolved for N2OR. Eight copper lines at 2.7 GHz, about nine lines at 4.6 GHz and about six lines at 9.2 GHz were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven-line patterns is proposed. These simulations indicated that Cu-Cu interaction, in a mixed-valence [Cu(1.5)...Cu(1.5)], S = 1/2 site is consistent with, if not proof of, the unusual spectral features observed for N2OR and COX. [ABSTRACT FROM AUTHOR]

A study is presented on the H+/e- stoichiometry for proton translocation by the isolated cytochrome bc1 complex under level-flow and steady-state conditions. An experimental procedure was used which allows the determination of pure vectorial proton translocation in both conditions in a single experiment. The results obtained indicate an H+/e- ratio of 1 at level-flow and 0.3 at steady-state. The ratios appear to be independent of the rate of electron transfer through the complex. Making use of pyranine-entrapped bc1 vesicles, a respiration-dependent steady-state ΔpH value of 0.4 was determined in the presence of valinomycin. This value could be either decreased by sub- saturating concentrations of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) or increased by introducing bovine serum albumin in the assay mixture. The steady-state H+/e- ratio appeared to be in linear inverse correlation with the ΔpH. This indicates that ΔpH exerts a control on the proton pump of the bc1 complex at the steady state. The effect of valinomycin-mediated potassium-diffusion potential on electron-transfer and proton- translocation activities is also shown. The experiments presented show that the H+/e- ratio is unaffected, both at level flow and steady state, by an imposed diffusion potential up to around 100 mV. At higher potential values the level-flow H+/e- ratio slightly decreased. Measurements as a function of imposed membrane potential of the rate of electron transfer at level flow and of the rate of the pre-steady-state reduction of b and c1 cytochromes in the complex indicate activation of electron transfer at potential values of 40–50 mV. This activation appears, however, to involve a rate-limiting step which remains normally coupled to proton translocation. [ABSTRACT FROM AUTHOR]

The haem-core architecture in cytochrome c3 isolated from Desulfovibrio baculatus (Norway 4) was probed using two-dimensional 1H NMR. Interhaem connectivities detected in NOE spectroscopy experiments performed at short mixing times are incompatible with the structure of the protein determined by X-ray crystallography, but agree instead with the haem arrangement found in cytochrome c3 from Desulfovibrio vulgaris (Miyazaki). These experiments show unequivocally that the relative orientation of the four haems in the two proteins is the same and does not invofve the 180° rotation of haems I and IV indicated in the X-ray structure determined for the cytochrome c3 from D. baculatus (Norway 4). [ABSTRACT FROM AUTHOR]

A series of nitrophenolic electron-transport inhibitors (2-substituted 4,6-dinitrophenols) of rat liver mitochondrial cytochrome-bc1 complex and of photosystem II (QB site) of spinach thylakoids was synthesized. The structure/inhibitory-activity relationship was examined to elucidate differences in the three-dimensional structure of the quinone redox site in the two systems. These inhibitors occupy the ubiquinone redox site of cytochrome-bc1 complex competitively with natural ubiquinol, probably at a Qo reaction center. The inhibitory activity tended to increase with the length of the 2- substituent, which may correspond to the isoprenoid side chain of ubiquinone and plastoquinone, increased in both experimental systems. However, the strict structural requirements of the 2-substitu- ent for binding to the ubiquinone or plastoquinone redox site were not identical. The alkyl substituents with a branching structure at the α-position to the benzene ring were favorable for inhibition of the cytochrome-bc1 complex, but not of photosystem II. Molecular-orbital calculations indicated that the main chain of 2-substituents with an α-branching structure was almost perpendicular to the benzene-ring plane because of steric congestion between the α-methyl and phenolic OH groups. The main chain of 2-substituents without an α-branching structure was flexible. Molecular-orbital studies indicated that ubiquinol was most stable when the portion of the isoprenoid side chain adjacent to the quinol ring was perpendicular to the quinol-ring plane, because of steric congestion by the vicinal OH and methyl groups. The side chain of plastoquinol was flexible because of the lack of a vicinal methyl group. Thus, the difference in the inhibitory activities between the two systems seemed to reflect the difference in the configuration of the isoprenoid side chain of ubiquinone and plastoquinone. These results suggested that the quinone redox site of the cytochrome-bc1 complex may recognize the configuration of the side chain near the quinone ring in the strict sense, whereas that of photosystem II (QB site) may recognize it in a loose sense. [ABSTRACT FROM AUTHOR]

Cytochrome P-450 species (P-450) comprise a polymorphic multigene family of heme-containing enzymes which are essential to the phase-I metabolism of xenobiotics. Induction of P-450 species by drugs and carcinogens has been extensively studied; endogenous regulation of P-450 also occurs during normal development and disease. The aim of this project was to study the in-vitro induction of P-450 and its modulation by inflammatory mediators in the human lung tumor-derived cell lines NCI H322 and NCI H358. The cell lines expressed detectable levels of 7-ethoxyresorufin O-deethylase which could be induced by benzanthracene. After benzanthracene treatment, a protein tentatively identified as isozyme CYP1A1 was detected by Western-blot analysis and a concommitant increase in CYP1A mRNA expression was observed. Optimal induction was observed at a benzanthracene concentration of 5 μg/ml with cells grown in RPMI medium containing 10% fetal calf serum. The effects of endotoxin, dexamethasone and five recombinant DNA-derived cytokines, interleukin-1β, tumor necrosis factor, and interferons α, β and γ, on constitutive and benzanthracene-induced ethoxyresorufin O-deethylase activity were examined in NCI H322 cells. Of all the lymphokines studied, only interferon γ had any marked effect. Administration of this lymphokine strongly suppressed ethoxyresorufin O-deethylase activity in both control and benzanthracene-treated cells. [ABSTRACT FROM AUTHOR]

Methylophilus methylotrophus cytochrome c″ is an unusual monohaem protein (15kDa) undergoing a redox-linked spin-state transition [Santos, H. & Turner, D. L. (1988) Biochim. Biophys. Acta 954, 277–286]. The midpoint redox potential of cytochrome c″ was measured over the pH range 4–10. The pH dependence of the midpoint redox potential was interpreted in terms of a model that considers the redox-state dependence of the ionization of two distinct and non-interacting protonated groups in the protein. This analysis led to the following pKa values within the pH range studied: pKa1o = 6.4, pKa1r = 5.4 and pKa2r = 8.1. Proton-NMR spectroscopy was used to assist the characterization of the two ionizing groups responsible for the observed redox-Bohr effect: the group ionizing with a lower pKar was assigned to a haem propionic acid substituent and the other to the axial histidine ligand which becomes detached upon reduction, which has a pKao too low to be measured. It is shown that M. methylotrophus cytochrome c″ is able to couple electron and proton transfer in the physiological pH range through a mechanism involving reversible change in the haem-iron coordination. Possible implications for the physiological role of the protein are discussed. [ABSTRACT FROM AUTHOR]

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Qo center (Q, ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The K of the G137R mutant was not affected and the Vmax was 50% higher. Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDS/PAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Qo center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit. [ABSTRACT FROM AUTHOR]

The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short-circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborought) and from Megasphaera elsdenii were applied. The observed pH values in reversed micelles were not dependent on the type of hydrogenase. Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sulfosuccinate types of reversed micelles were used in combination with viologens having distinguishable valencies. It was observed that, in the cationic-reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic-reversed micelles. Furthermore, the dissociation constant was independent of the concentration of viologen semiquinone in the reversed micelle, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a · pHrm + b, between the pH of the bulk water and the pH as measured in the revered micelle (pHrm). In all these cases the value of α is smaller than unity, the value of b ranges between 1.6–2.7. For DAP the pHrm is always around 7. In DAP-reversed micelles, the counter-ion propionate probably serves as an internal buffer. Using cytochrome c3 as pH indicator in combination with N,N′-di(3-aminopropyl)-4,4′-bipyridinium)4+ to take care of electron transfer, in cetyltrimethylammonium-bromide-reversed micelles the pHrm is about the same as indicated by the viologen; in SDS-reversed micelles the pHrm is always lower than that indicated by N,N′-di(3-aminopropyl)-4,4′-pyridinium4+. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. vulgaris hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly. [ABSTRACT FROM AUTHOR]

We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P4501VA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P4501VA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P4501VA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region. [ABSTRACT FROM AUTHOR]

The regional expression of six different cytochrome P450 (CYP) forms in rat liver under constitutive and induced conditions was compared using immunological techniques, lmmunostaining of consecutive thin sections from control liver revealed that the same hepatocytes, forming a 6-8 cells thick layer surrounding the terminal hepatic venules, were stained for CYP2B1/2, CYP2EI and CYP3A1. Staining of CYP2A1 extended further into the midzonal region, whereas all cells of the acinus stained for CYPEtOH2, These results were supported by Western blot analysis of cell lysates from the periportal or perivenous region obtained by zone-restricted digitonin treatment during in situ perfusion. The data suggest three distinct patterns of constitutive P450 expression: perivenousrestricted (CYP2BI/2, CYP2E1 and CYP3A1); pefivenous-dominated (CYP2AI) and panacinar (CYPEtOH2). Chronic exposure to ethanol caused induction of CYP2E1 in the same cells already being constitutively expressed, whereas CYPEtOH2 was more induced in the pefiportal area. The relative induction of CYP2B1/2, CYP3A1 and CYPEtOH2 after treatment with phenobarbital was stronger in periportal hepatocytes, resulting in levelling out of the initial pedvenous dominance of CYP2B1/2 and CYP3A1, whereas CYPEtOH2 became pefiportal-dominated. Acetone induced CYP2E1, CYP2Cll and CYP3A1 selectively in the perivenous area. These studies indicate that a particular P450 isozyme is generally induced in the same cells where it is constitutively expressed, and that this regional selectivity is independent of the kind of inducer. The data suggest that, during maturation, the hepatocytes acquire various phenotypes in the pefiportal and perivenous region, to respond differently to endogenous and exogenous signals in the control of P450 expression. [ABSTRACT FROM AUTHOR]

An NMR study of the cytochrome c variant Asn52Ile is used to show how the redox state change in native cytochrome c is coupled to a rearrangement of a proton network which runs through the cytochrome c molecule. The substitution breaks the H-bond network and removes the coupling. The uncovering of this putative proton channel and the connection of changes to it with redox state changes of the iron centre of the protein allows a possible description of the way in which redox energy state changes can be coupled to energisation and gating of protons in membranes. [ABSTRACT FROM AUTHOR]

tH two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatium vinosum have been obtained. The protein is of medium size (Mr 28000), essentially high spin (S = 5/2) although some quantum mechanical spin admixing with S = 3/2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (α-CH2 propionate and β-CH2 protons of the bound histidine) and between α-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems. [ABSTRACT FROM AUTHOR]

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish­Bowden, A. & Koshland, D. E. Jr (1970) J. Biol. Chem. 245, 6241–6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J. J. & Xavier, A. V. (1991) Eur. J. Biochem. 202, 1101–1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network. [ABSTRACT FROM AUTHOR]

A thermodynamic model is presented to describe the redox behaviour of the tetraheme cytochrome c3 from Desulfovibrio gigas. This molecule displays different intrinsic redox potentials for the four hemes and during the redox titration process, interactions among different hemes occur, thus altering the values of redox potentials according to which of the hemes are oxidized [Santos, H., Moura, J. J. G., Moura, I., LeGall, J. & Xavier, A. V. (1984) Eur. J. Biochem. 141, 283–296]. This complex cooperative behaviour [Xavier, A. V. (1986) J. Inorg. Biochem. 28, 239–243] has been analyzed here using an I2H4-interaction network [Cornish­Bowden, A. & Koshland, D. E. Jr (1970) J. Biol. Chem. 245, 6241–6250] coupled to a proton-linked equilibrium between two tertiary structures. Such a formalism, which requires a reduced number of parameters, is able to fully account quantitatively for the pH dependence of the NMR redox-titration curves. The ‘redox-Bohr’ effect is discussed in terms of the available structure and thermodynamic data and a functional mechanism is proposed. [ABSTRACT FROM AUTHOR]

An internal water molecule (designated WAT166) is found in iso-1-cytochrome c which is part of a redox-state-dependent hydrogen bond network. The position of this water molecule with respect to the polypeptide fold can altered or even displaced by site-directed mutagenesis leading to structural perturbations and associated changes m redox potential. Using saturation transfer 1H-NMR methods, this study measures changes in the electron transfer reactivity for three variants of yeast iso-1-cytochromes c in which the position of this water molecule is altered. In particular, the reverse electron transfer rate is measured within a complex formed between either wild-type or variant yeast iso-1-cytochromes c and the tryptic fragment of bovine liver microsomal cytochrome bs. For three variants of yeast iso-1-cytochrome c the rate constants measured by saturation transfer are wild-type (Ash52, Eo = 270 mV, kex = 0.3 s-1), Ash52 → Ala (Eo = 240 mV, kex = 0.6 s-1), Ash52 → Ile (Eo = 220 mV, kex = 1.0 s-1). The first-order fates ate compared with that of a fourth variant Phe82 → Gly which has been measured previously (Eo = 220 mV, kex = 0.7 s-1). An analysis of the variation in the observed cross exchange rote using Marcus theory shows that these changes can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome c, surprisingly it does not significantly influence the intrinsic electron transfer reactivity of the protein. Studies of the activation parameters suggests that a variation of temperature changes both ΔG∗ and also the prefactor. These data are discussed in terms of models involving dynamic molecular recognition between proteins. [ABSTRACT FROM AUTHOR]

The heme components of chlorosome-depleted membranes of the green-gliding bacterium Chloroflexus aurantiacus were studied by EPR spectroscopy. The four major species, which are present in approximately equimolar quantities, are characterized by the following gz values, redox midpoint potentials and orientations of heme planes with respect to the plane of the membrane: gz = 3.40, Em= + 280 mV, 30°; gz = 3.33, Em= 0 mV, 45°; gz = 3.03, Em = +95 mV, 40-50° and gz = 2.95, Em= + 150 mV, 90°. These four hemes were attributed to cytochrome c554, the membrane-bound immediate electron donor to the photosynthetic reaction centre in Chloroflexus. All hemes except that with the highest potential were able to undergo photooxidation at 4 K. The photooxidation of the lowest potential heme was stable, whereas that of the + 95 mV and the + 150 mV hemes reversed on increasing the temperature to 100 K in darkness, due to charge recombination. The ability to photooxidize these heroes at 4 K was lost upon aging of samples. The results demonstrate that a reaction-centre-associated tetraheme cytochrome subunit, analogous to that of purple bacteria, is also present in C. aurantiacus. [ABSTRACT FROM AUTHOR]