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 mu M, k(cat) 3960 min(-1); Y51F mutant, K-m 432 mu M, k(cat) 6140 min(-1); wild-type, K-m 288 mu M, k(cat) 5140 min(-1)). A slightly increased k(cat) for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (Delta G double dagger) resulting from a smaller Delta G 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 mu M, k(cat) 14620 min(-1); compared with values of 4.7 mu M 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 mon tightly than wild-type.