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 (K_d 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 (k_lim = 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 k_lim 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.

Key Words :

• Cytochrome P450
• Cytochrome b₅
• Enzyme mechanism
• Electron transfer
• P450 BM3