Analysis of the Interactions of Cytochrome b5 with Flavocytochrome P450 BM3 and its Domains

Abstract

Interactions between a soluble form of microsomal cytochrome b5 (b5) from Musca domestica (housefly) and Bacillus megaterium flavocytochrome P450 BM3 and its component reductase (CPR), heme (P450) and FAD/NADPH-binding (FAD) domains were analyzed by a combination of steady-state and stopped-flow kinetics methods, and optical spectroscopy techniques. The high affinity binding of b5 to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (Kd for b5 binding = 0.44 μM and 0.72 μM for the heme domain and intact flavocytochrome, respectively). The b5 had modest inhibitory effects on steady-state turnover of P450 BM3 with fatty acids, and the ferrous-carbon monoxy P450 complex was substantially stabilized on binding b5. Single turnover reduction of b5 by BM3 using stopped-flow absorption spectroscopy (klim = 116 s−1) was substantially faster than steady-state reduction of b5 by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b5 heme electron transfer in the steady-state reaction. Steady-state b5 reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the klim for b5 reduction ∼10-fold, and also resulted in a lag phase in steady-state b5 reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b5 could not reduce the ferric P450 BM3 heme domain under anaerobic conditions, consistent with heme iron reduction potentials of the two proteins. However, rapid oxidation of both hemoproteins occurred on aeration of the ferrous protein mixture (and despite the much slower autoxidation rate of b5 in isolation), consistent with electron transfer occurring from b5 to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b5 and a model prokaryotic P450. Binding of b5 perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b5 interactions with eukaryotic P450s. These results are consistent with the conservation of structure of P450s (particularly at the heme proximal face) between prokaryotes and eukaryotes, and may point to as yet undiscovered roles for b5-like proteins in the control of activities of certain prokaryotic P450s.