Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4.

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E(0')=-450 mV) and electrodes, modified with cytochrome c (E(0')=-50 mV) or Prussian Blue (E(0')=0), as measuring electrodes (for H(2)O(2)) and Clark-type electrode (for O(2)). Benzphetamine N-demethylation rate was 17+/-3 nmol/nmol of enzyme/min, peroxide production was 4.8+/-0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3+/-0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by capital P450 2B4 was 19.4+/-0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8+/-0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.