The Role of NADH in Uncoupled Microsomal Monoxygenations

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Abstract
The stoichiometry of the liver microsomal monoxygenase system from phenobarbital‐pretreated rabbits has been investigated with cyclohexane, n‐hexane and perfluoro‐n‐hexane as substrates. With cyclohexane an almost 1:1:1 stoichiometry for cyclohexanol: NADPH: O2 was obtained. With the uncoupler perfluoro‐n‐hexane no product was formed and 2 moles of NADPH were oxidised per mole of oxygen. The stoichiometry of n‐hexane hydroxylation suggested that partial uncoupling with this substrate had occurred. NADH addition together with NADPH had no significant effect on the hydroxylation of cyclohexane but led to a more than additive hydroxylation in the case of n‐hexane. Oxygen uptake with perfluoro‐n‐hexane and NADPH was almost doubled in the presence of NADH. It was shown that during uncoupling an increased oxidation of NADH via cytochrome b5 occurs. It was concluded that active oxygen which is not used for monoxygenations is reduced to water by the NADH‐cytochrome b5 system and that this sparing effect could be the main mechanism of the well‐known synergistic action of NADH on microsomal hydroxylations.