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

  • Perret A
  • Pompon D
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Abstract

Contradictory mechanisms involving conformational or redox effects have been proposed for the enhancement of cytochrome P450 activities by cytochrome b5 in reconstituted systems. These mechanisms were reinvestigated for human liver P450 3A4 bound to recombinant yeast membranes including human P450 reductase and various levels of human b5. 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 b5. P450 uncoupling by hydrogen peroxide formation was decreased by b5, leading to a corresponding increase in the rate of ferryl-oxo complex formation. Nevertheless, the major b5 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 b5 were strictly required for this modulation and that redox state of b5 was ruled by the nature and concentration of 3A4 substrates. Moreover, rapid kinetic analysis of b5 reduction following NADPH addition suggested that b5 was reduced by the 3A4 ferrous-dioxygen complex and reoxidized by subsequent P450 oxygenated intermediates. A kinetic model involving a 3A4-b5 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 b5 analogue previously reported in reconstituted systems. In this model, synchronization of the b5 and 3A4 redox cycles, binding site overlap between b5 and reductase, and dynamics of the b5-3A4 complex were critical features. This model opened the way for designing complementary experiments for unification of b5 action mechanisms on P450s.

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Authors

  • Alain Perret

  • Denis Pompon

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