Switching Pyridine Nucleotide Specificity in P450 BM3

Abstract

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...

Engineering Cytochrome P450 Enzymes for Improved Activity towards Biomimetic 1,4‐NADH Cofactors

“…These results clearly demonstrate that protein engineering can be used to generate a mutant of P450 BM-3 that has good activity with at least two NADH biomimics toward which the wild-type enzyme was inactive. Although P450 BM-3 is a good model system for reductase proteins that have both an FAD and FMN ligand, such as human P450 reductase, [11] the reductase of P450cam is a more appropriate model for other oxygenase-coupled NADH-dependent ferredoxin reductases (ONFRs) that are necessary for electron transport to a number of hydroxylases, including alkane and benzene/diphenyl hydroxylases. [12] Unlike wild-type (WT) P450 BM-3, WT P450cam was able to catalyze substrate oxidation by using both 1 a and 1 b (Table 3).…”

C-terminal Tail Residue Arg1400 Enables NADPH to Regulate Electron Transfer in Neuronal Nitric-oxide Synthase

“…Phe 1395 lies just before the start of the C-terminal tail, and its side-chain phenyl group is expected to undergo significant movement upon NADP(H) binding so that a nicotinamide-FAD stacking interaction can occur that is required for hydride transfer (22,24,27). Previous results suggest that the Phe 1395 side chain is important for regulating the conformational equilibrium of the FMN module in nNOSr, because Phe 1395 must be present to stabilize the FMN-shielded conformation and repress electron transfer when NADP(H) binds (33,34,65).…”

“…NOSr contains separate ferredoxin-NADP ϩ -reductase (FNR) and FMN modules (21,22) and in this way is similar to a number of NADPH-utilizing dual flavin oxidoreductases (23)(24)(25)(26)(27). In NOSr, a hydride transfer occurs from NADPH to FAD within the FNR module, followed by electron transfer from the FAD hydroquinone to FMN (17,28,29).…”

Structural and Spectroscopic Characterization of P450 BM3 Mutants with Unprecedented P450 Heme Iron Ligand Sets

“…Steady-state kinetic analysis was carried out with WT, A264K, and A264H flavocytochromes as described previously (21), using laurate and arachidonate as substrates to monitor substrate-dependent NADPH oxidation. Reductasedependent cytochrome c reduction was also monitored for WT and mutants as described previously (8,23). Reduction of P450s and binding of carbon monoxide was performed as described previously (6).…”