Electron Transfer in Flavocytochrome P450 BM3:  Kinetics of Flavin Reduction and Oxidation, the Role of Cysteine 999, and Relationships with Mammalian Cytochrome P450 Reductase

Roitel, Olivier; Scrutton, Nigel S.; Munro, Andrew W.

doi:10.1021/bi034562h

Cited by 45 publications

(76 citation statements)

References 35 publications

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“…SDS-PAGE (15%) indicated that the proteins were pure. The concentration of purified proteins was measured spectrophotometrically using molar extinction coefficients 11,300 M Ϫ1 cm Ϫ1 for the FAD domains and 21,200 M Ϫ1 cm Ϫ1 for the reductase domains, as described previously (7,31,32).…”

Section: Methodsmentioning

confidence: 99%

“…Absorption transients from the stopped-flow experiments were fitted to appropriate exponential functions using Spectrakinetics software (Applied Photophysics). Observed rate constants for flavin reduction by NADH showed a hyperbolic dependence on coenzyme concentration, and data were fitted to Equation 1 (31) to determine the apparent enzyme-coenzyme dissociation constant, K, and the limiting rate of hydride transfer (k lim ).…”

Section: Methodsmentioning

confidence: 99%

“…For the redox titrations of the W1046A and W1046H reductase domains, data were fitted to Equation 3, describing a 4-electron redox process, as described in previous studies (6,10). To enable the fitting process, data for the redox couples of the FMN from previous studies were fixed in the initial fitting regime (31). The restraints were then released, and subsequent iteration provided potential values close to those obtained from the initial estimate.…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies on wild-type domains have shown that the affinity resin 2Ј,5Ј-ADP-agarose can be used to purify the NADPH-binding proteins (7,31,32). However, the mutant FAD and diflavin reductase proteins bound only weakly to this resin.…”

Section: Expression and Purification Of Wild-type And Trp-1046 Mutantmentioning

confidence: 99%

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Switching Pyridine Nucleotide Specificity in P450 BM3

Neeli

Roitel²,

Scrutton

et al. 2005

Journal of Biological Chemistry

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Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k cat /K m (NADPH)/k cat /K m (NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wildtype reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a chargetransfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH 2 -NAD(P) ؉ charge-transfer species, suggesting a relatively slow rate of release of NAD(P)؉ from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/ semiquinone couple, and a concomitant ϳ100-mV elevation in the 2-electron redox couple for the enzymebound FAD (؊320, ؊220, and ؊224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).Bacillus megaterium flavocytochrome P450 BM3 is a high activity fatty acid hydroxylase enzyme that has evolved from fusion of a eukaryotic-like NADPH-cytochrome P450 reductase (CPR) 1 to a P450 in a single polypeptide (1). The fusion provides an efficient electron transport system from NADPH, through the FAD and FMN cofactors in the CPR, and onto the heme b in the P450 (2). P450 BM3 has the highest monooxygenase activity of any P450 system examined to date (3). Molecular dissection has been used to generate the individual heme and CPR domains for structural, thermodynamic, and kinetic studies (e.g. Refs. 4 -7). The component flavodoxin-like (FMN-containing) and ferredoxin reductase-like (FAD-containing) domains of the CPR have also been generated, supporting the hypothesis that the enzyme has a discrete domain organization and that the BM3 CPR and other related enzymes evolved by fusion of genes encoding the smaller flavoproteins (8). Molecular dissection of the related human CPR to produce component flavin-binding domains has been reported (9). The isolated domains of human CPR exchange electrons and have thermodynamic properties almost identical to those of intact CPR (9, 10). The availability of isolated domains has enabled detailed study of electron transfer mechanisms in wild-type and mutant proteins (11,12).Fla...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Expression and Purification Of Wild-type And Trp-1046 Mutantmentioning

confidence: 99%

See 2 more Smart Citations

Switching Pyridine Nucleotide Specificity in P450 BM3

Neeli

Roitel²,

Scrutton

et al. 2005

Journal of Biological Chemistry

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“…These residues are represented in rat nNOS by Asp-1393, Ser-1176, and Cys-1349 (27). Mutagenic substitution studies on the analogously positioned residues in FNR enzymes, cytochrome P450 reductase, and other related enzymes have established that each of the three residues helps to enhance the rate of hydride transfer between NAD(P)H and FAD (32)(33)(34)(35)(36)(37)(38)(39)(40). Given the structural and regulatory properties of the NOSs, we sought to examine how one of these conserved residues (Asp-1393, Fig.…”

Section: Nitric Oxide (No)mentioning

confidence: 99%

A Conserved Aspartate (Asp-1393) Regulates NADPH Reduction of Neuronal Nitric-oxide Synthase

Panda¹,

Adak²,

Konas

et al. 2004

Journal of Biological Chemistry

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Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP ؉ reductase (FNR)-like module that contains NADPH-and FADbinding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaMdependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPHdependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS. Nitric oxide (NO)1 is a widespread signal and effector molecule in biology (1-3). Animals generate NO by virtue of the nitric-oxide synthases (NOSs), which catalyze a stepwise, NADPH-dependent oxidation of L-Arg to NO and L-citrulline, with N -hydroxy-L-arginine (NOHA) being formed as an intermediate. Each NOS is composed of an N-terminal oxygenase domain that binds iron protoporphyrin IX (heme), (6R)-tetrahydrobiopterin (H 4 B), and substrate, L-Arg, and a C-terminal flavoprotein domain that binds FAD, FMN, and NADPH. A calmodulin (CaM)-binding sequence connects the oxygenase and flavoprotein domains. During catalysis, electrons from NADPH transfer into the FAD and FMN groups of the NOS flavoprotein domain, and then transfer one at a time from the FMN hydroquinone (FMNH 2 ) to the ferric heme (4 -8) (Scheme 1).Heme reduction enables the enzyme to bind O 2 and initiate an H 4 B-dependent oxygen activation that is required for catalysis (9, 10). In all NOSs studied so far, the rates of NADPHdependent flavin reduction are fast relative to the rates of electron transfer from FMNH 2 to the ferric heme in the CaMbound enzymes (11)(12)(13)(14). This makes heme reduction rate-limiting for NO synthesis and implies that the NOS flavins predominantly exist in their reduced forms during steady-state NO synthesis.NOSs are part of a small enzyme family whose members have covalently linked flavoprotein and heme domain...

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