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Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.

Midpoint reduction potentials for the flavin cofactors in human NADPH-cytochrome P450 oxidoreductase were determined by anaerobic redox titration of the diflavin (FAD and FMN) enzyme and by separate titrations of its isolated FAD/NADPH and FMN domains. Flavin reduction potentials are similar in the isolated domains (FAD domain E(1) [oxidized/semiquinone] = -286 +/- 6 mV, E(2) [semiquinone/reduced] = -371 +/- 7 mV; FMN domain E(1) = -43 +/- 7 mV, E(2) = -280 +/- 8 mV) and the soluble diflavin reductase (E(1) [FMN] = -66 +/- 8 mV, E(2) [FMN] = -269 +/- 10 mV; E(1) [FAD] = -283 +/- 5 mV, E(2) [FAD] = -382 +/- 8 mV). The lack of perturbation of the individual flavin potentials in the FAD and FMN domains indicates that the flavins are located in discrete environments and that these environments are not significantly disrupted by genetic dissection of the domains. Each flavin titrates through a blue semiquinone state, with the FMN semiquinone being most intense due to larger separation (approximately 200 mV) of its two couples. Both the FMN domain and the soluble reductase are purified in partially reduced, colored form from the Escherichia coli expression system, either as a green reductase or a gray-blue FMN domain. In both cases, large amounts of the higher potential FMN are in the semiquinone form. The redox properties of human cytochrome P450 reductase (CPR) are similar to those reported for rabbit CPR and the reductase domain of neuronal nitric oxide synthase. However, they differ markedly from those of yeast and bacterial CPRs, pointing to an important evolutionary difference in electronic regulation of these enzymes.

Neuronal nitric-oxide synthase (nNOS) is activated by the Ca 2؉ -dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Presteady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP ؉ release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the fulllength enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.Mammalian nitric-oxide synthases (NOS) 1 are responsible for generating NO in a wide range of cell types during the immune system response and as part of numerous intercellular signaling mechanisms (1-4). They are homodimeric and consist of a reductase domain, which binds FAD and FMN stoichiometrically, and an oxygenase domain, which contains a P450-like Cys-ligated heme and a tetrahydrobiopterin molecule. The oxygenase domain forms the main dimer interface, and tetrahydrobiopterin is an integral part of this. Crystal structures are available for several NOS oxygenase domain dimers (5, 6) and for the FAD binding subdomain of neuronal NOS (nNOS) (7). The reductase domain closely resembles mammalian cytochrome P450 reductase (7-11) and similarly catalyzes NADPH dehydrogenation at the FAD site and electron transfer to the FMN. The oxygenase domain of one subunit accepts electrons from the reductase domain of the other subunit (12-14) and generates NO from L-arginine via a unique two-step monooxygenation reaction (1-4, 15). The two domains are linked by a functional peptide of 20 -25 amino acids which binds calmodulin (CaM) reversibly at elevated Ca 2ϩ concentrations in the nNOS and endothelial NOS (eNOS) isoforms but irreversibly in the inducible isoform (iNOS). CaM binding activates nNOS and eNOS, providing them with a rapid response mechanism during their participation in signaling cascades. The inducible isoform, on the other hand, is regulated at the transcriptional level. CaM binding has been shown to control NO synthesis by activating electron transfer th...

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The neuronal and endothelial nitric-oxide synthases (nNOS and eNOS) differ from inducible NOS in their

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.

Rapid events in the processes of electron transfer and substrate binding to cytochrome P-450 BM3 from Bacillus megaterium and its constituent haem-containing and flavin-containing domains have been investigated using stopped-flow spectrophotometry. The formation of a blue semiquinone flavin form occurs during the NADPH-dependent reduction of the flavin domain and a species with a similar absorption maximum is also seen during reduction of the holoenzyme by NADPH. EPR spectroscopy confirms the formation of the flavin semiquinone. The formation of this semiquinone is transient during fatty acid monooxygenation by the holoenzyme, but in the presence of excess NADPH the species reforms once fatty acid is exhausted. Electron transfers through the reductase domain are too rapid to limit the fatty acid monooxygenation reaction. The substrate-binding-induced haem iron spin-state shift also occurs much faster than the k,,, at 25°C. The rate of first electron transfer to the haem domain is also rapid; but it is of the order of 5-10-times larger than the k,,, for the enzyme (dependent on the fatty acid used).Given that two successive electron transfers to haem iron are required for the oxygenation reaction, these rates are likely to exert some control over the rate of fatty acid oxygenation reactions. The presence of large amounts of NADPH also results in decreased rates of electron transfer from flavin to haem iron. In the difference spectrum of the active fatty acid hydroxylase, features indicative of a high-spin iron haem accumulate. These are in accordance with the presence of large amounts of an Fe'+-product bound enzyme during turnover and indicate that product release may also contribute to rate limitation. Taken together, these data suggest that the catalytic rate is not determined by the accumulation of a single intermediate in the reaction scheme, but rather that it is controlled in a series of steps.Keywords: cytochrome P-450; stopped-flow kinetics ; EPR; electron transfer.The cytochrome P-450 monooxygenases (P-450) are a ubiquitous superfamily of haem enzymes which catalyse insertion of oxygen into an enormous variety of both physiological and nonphysiological organic substrates [l -31. P-450 generally fall into one of two broad classes. Class I P-450 (bacterial/mitochondrial) are three component systems comprised of an NAD(P)H-binding flavoprotein reductase, a small iron-sulfur protein and the P-450, which is membrane bound in eukaryotic forms [4]. Class I1 P-450 (microsomal) are two component systems comprising an FAD-containing and FMN-containing NADPH-cytochrome P-450 reductase and the P-450. This class is found almost exclusively in eukaryotes, where both components are membrane bound [4].There are many prokaryotic class I P-450, the best characterised being the P-450cum camphor hydroxylase from Pseudomonus putida. P-450cum (CUP 101) was the first P-450 for which an atomic structure was determined [5]. More recently, a unique prokaryotic class I1 flavocytochrome P-450 from Bacillus megaterium has been char...