Electron shuttle between membrane-bound cytochrome P450 3A4 and b5 rules uncoupling mechanisms

Contradictory mechanisms involving conformational or redox effects have been proposed for the enhancement of cytochrome P450 activities by cytochrome b(5) in reconstituted systems. These mechanisms were reinvestigated for human Liver P450 3A4 bound to recombinant yeast membranes including human P450 reductase and various levers of human b(5), Species conversions were calculated on the basis of substrate, oxygen, and electronic balances in six different substrate conditions. Electron flow from P450 reductase to ferric 3A4, was highly dependent on the nature of substrate but not on the presence of b(5), P450 uncoupling by hydrogen peroxide formation was decreased by b(5), leading to a corresponding increase in the rate of ferryl-oxo complex formation. Nevertheless, the major b(5) effects mainly relied on an increased partition of ferryl-oxo complex to substrate oxidation compared to reduction to water, which could support a conformation change based mechanism. However, further steady-state investigations evidenced that electron carrier properties of b(5) were strictly required for this modulation and that redox state of b(5) was ruled by the nature and concentration of 3A4 substrates. Moreover, rapid kinetic analysis of b(5) reduction following NADPH addition suggested that b(5) was reduced by the 3A4 ferrous-dioxygen complex and reoxidized by subsequent P450 oxygenated intermediates. A kinetic model involving a 3A4-b(5) electron shuttle within a single productive P450 cycle was designed and adjusted. This model semiquantitatively simulated all presented experimental data and can be made compatible with the effect of the redox-inactive b(5) analogue previously reported in reconstituted systems. In this model, synchronization of the bs and 3A4 redox cycles, binding site overlap between b(5) and reductase, and dynamics of the b(5)-3A4 complex were critical features. This model opened the way for designing complementary experiments for unification of b(5) action mechanisms on P450s.