Interactions between a soluble form of microsomal cytochrome b(5) (b(5)) 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 b(5) to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (K(d) for b(5) binding = 0.44 microM and 0.72 microM for the heme domain and intact flavocytochrome, respectively). The b(5) 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 b(5). Single turnover reduction of b(5) by BM3 using stopped-flow absorption spectroscopy (k(lim) = 116 s(-1)) was substantially faster than steady-state reduction of b(5) by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b(5) heme electron transfer in the steady-state reaction. Steady-state b(5) reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the k(lim) for b(5) reduction approximately 10-fold, and also resulted in a lag phase in steady-state b(5) reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b(5) 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 b(5) in isolation), consistent with electron transfer occurring from b(5) to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b(5) and a model prokaryotic P450. Binding of b(5) perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b(5) 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 b(5)-like proteins in the control of activities of certain prokaryotic P450s.