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 kcat 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 kcat 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 Fe3+-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.
cytochrome P-450 monooxygenase