Mechanistic studies on the reductive half-reaction of NADPH-cytochrome P450 oxidoreductase.
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Site-directed mutagenesis has been employed to study the mechanism of hydride transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specifically, Ser457, Asp675, and Cys630 have been selected because of their proximity to the isoalloxazine ring of FAD. Substitution of Asp675 with asparagine or valine decreased cytochrome c reductase activities 17- and 677-fold, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytochrome c reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A., and Kasper, C. B. (1996) Biochemistry 35, 9451-9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly multiplicative decreases in cytochrome c reductase activity (774- and 22000-fold, respectively) with corresponding decreases in the rates of flavin reduction. For each mutation, increases were observed in the magnitudes of the primary deuterium isotope effects with NADPD, consistent with decreased rates of hydride transfer from NADPH to FAD and an increase in the relative rate limitation of hydride transfer. Asp675 substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shifted the pKa of an ionizable group previously identified as necessary for catalysis (Sem, D. S., and Kasper, C. B. (1993) Biochemistry 32, 11539-11547) from 6.9 to 7.8. These results are consistent with a model in which Ser457, Asp675, and Cys630 stabilize the transition state for hydride transfer. Ser457 and Asp675 interact to stabilize both the transition state and the FAD semiquinone, while Cys630 interacts with the nicotinamide ring and the fully reduced FAD, functioning as a proton donor/acceptor to FAD. (+info)
The flavin environment in old yellow enzyme. An evaluation of insights from spectroscopic and artificial flavin studies.
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Spectroscopic and chemical modification studies of modified flavins bound to old yellow enzyme have led to predictions about the flavin environment of this enzyme. These studies analyzed solvent accessibility and hydrogen bonding patterns of particular flavin atoms, in addition to suggesting amino acid residues that are in close proximity to those atoms. Here, these studies are evaluated in the light of the crystal structure of old yellow enzyme to reveal that the spectroscopic and modified flavin results are generally consistent with the crystal structure. This highlights the fact that these are useful methods for studying flavin binding site structure. Although several of the inferred properties of the flavin environment are not consistent with the crystal structure, these discrepancies occurred in cases where an incorrect choice was made from among multiple plausible explanations for an experimental result. We conclude that modified flavin studies are powerful probes of flavin environment; however, it is risky to specify details of interactions, especially because of uncertainties due to induced charge delocalization in the flavin. (+info)
The identification of primary sites of superoxide and hydrogen peroxide formation in the aerobic respiratory chain and sulfite reductase complex of Escherichia coli.
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The fitness of organisms depends upon the rate at which they generate superoxide (O-2) and hydrogen peroxide (H2O2) as toxic by-products of aerobic metabolism. In Escherichia coli these oxidants arise primarily from the autoxidation of components of its respiratory chain. Inverted vesicles that were incubated with NADH generated O-2 and H2O2 at accelerated rates either when treated with cyanide or when devoid of quinones, implicating an NADH dehydrogenase as their source. Null mutations in the gene encoding NADH dehydrogenase II averted autoxidation of vesicles, and its overproduction accelerated it. Thus NADH dehydrogenase II but not NADH dehydrogenase I, respiratory quinones, or cytochrome oxidases formed substantial O-2 and H2O2. NADH dehydrogenase II that was purified from both wild-type and quinone-deficient cells generated approximately 130 H2O2 and 15 O-2 min-1 by autoxidation of its reduced FAD cofactor. Sulfite reductase is a second autoxidizable electron transport chain of E. coli, containing FAD, FMN, [4Fe-4S], and siroheme moieties. Purified flavoprotein that contained only the FAD and FMN cofactors had about the same oxidation turnover number as did the holoenzyme, 7 min-1 FAD-1. Oxidase activity was largely lost upon FMN removal. Thus the autoxidation of sulfite reductase, like that of the respiratory chain, occurs primarily by autoxidation of an exposed flavin cofactor. Great variability in the oxidation turnover numbers of these and other flavoproteins suggests that endogenous oxidants will be predominantly formed by only a few oxidizable enzymes. Thus the degree of oxidative stress in a cell may depend upon the titer of such enzymes and accordingly may vary with growth conditions and among different cell types. Furthermore, the chemical nature of these reactions was manifested by their acceleration at high temperatures and oxygen concentrations. Thus these environmental parameters may also directly affect the O-2 and H2O2 loads that organisms must bear. (+info)
Roles of key active-site residues in flavocytochrome P450 BM3.
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The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type. (+info)
Riboflavin and riboflavin-derived cofactors in adolescent girls with anorexia nervosa.
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BACKGROUND: Thyroid hormones, riboflavin, riboflavin cofactors, and organic acids were assessed in girls with anorexia nervosa. OBJECTIVE: The objective was to examine the effect of malnutrition and low thyroid hormone concentrations on erythrocyte and plasma riboflavin metabolism and their relation with urinary organic acid excretion. DESIGN: Seventeen adolescent girls with anorexia nervosa [body mass index (BMI; in kg/m2): 14.8 +/- 2.2] and 17 age-matched, healthy girls (control subjects; BMI: 20.5 +/- 2.2) took part in the feeding study. Erythrocyte and plasma riboflavin as well as riboflavin cofactors (flavin mononucleotide and flavin adenine dinucleotide) were assessed by HPLC, whereas urinary organic acids were assessed by gas chromatography-mass spectrometry. RESULTS: Anorectic patients who began a feeding program had higher erythrocyte riboflavin (3.5 +/- 2.2 compared with <0.1 nmol/mol hemoglobin; P < 0.001), lower plasma flavin adenine dinucleotide (57.8 +/- 18.5 compared with 78.5 +/- 54.3 nmol/L; P < 0.05), and higher urinary ethylmalonic acid (7.12 +/- 4.39 compared with 1.3 +/- 2.8 micromol/mmol creatinine; P < 0.001) and isovalerylglycine (7.65 +/- 4.78 compared with 3.8 +/- 0.9 micromol/mmol creatinine; P < 0.05) concentrations than did control subjects. Triiodothyronine concentrations were low and negatively correlated with plasma riboflavin concentrations (r = -0.69, P < 0.01). Not all patients showed improvements in these biochemical indexes after 30 d of refeeding. CONCLUSIONS: The low triiodothyronine concentrations observed in anorexia nervosa could alter the extent of riboflavin conversion into cofactors, thus leading to high erythrocyte riboflavin concentrations, low plasma flavin adenine dinucleotide concentrations, and high rates of ethylmalonic acid and isovalerylglycine excretion. (+info)
Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation.
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We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations. (+info)
Cytochrome c-dependent methacrylate reductase from Geobacter sulfurreducens AM-1.
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Geobacter sulfurreducens AM-1 can use methacrylate as a terminal electron acceptor for anaerobic respiration. In this paper, we report on the purification and properties of the periplasmic methacrylate reductase, and show that the enzyme is dependent on the presence of a periplasmic cytochrome c (apparent K(m) = 0.12 microM). The methacrylate reductase was found to be composed of only one polypeptide with an apparent molecular mass of 50 kDa and to contain, bound tightly but not covalently, 1 mol of FAD per mol. The N-terminal amino acid sequence showed sequence similarity to a periplasmic fumarate reductase from Shewanella putrefaciens. However, methacrylate reductase did not catalyze the reduction of fumarate. The periplasmic cytochrome c, which was also purified, had an apparent molecular mass of 30 kDa and contained approximately 4 mol of heme.mol(-1). Cells of G. sulfurreducens AM-1 grown on acetate and methacrylate as an energy source were found to contain all the enzymes required for the oxidation of acetate to CO(2) via the citric acid cycle. (+info)
Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.
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Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation. (+info)