Roles of key active-site residues in flavocytochrome P450 BM3

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, K(m) 859 μM, k(cat) 3960 min-1; Y51F mutant, K(m) 432 μM, k(cat) 6140 min-1; wild-type, K(m) 288 μM, k(cat) 5140 min-1). A slightly increased k(cat) for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG≠) resulting from a smaller ΔG 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, K(m) is massively increased and k(cat) is decreased for oxidation of both laurate (K(m) 2.08 mM, k(cat) 2450 min-1) and arachidonate (K(m) 34.9 μM, k(cat) 14620 min-1; compared with values of 4.7 μM 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 K(m) and decreased k(cat) 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: k(cat) 'fast', 760 (1620) min-1; k(cat) 'slow', 48.0 (44.6) min-1; k(conv) (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.