Diminished FAD‐Binding Specificity in Human Cytochrome P450 Oxidoreductase Associated with Antley‐Bixler Syndrome

Abstract

Antley-Bixler syndrome (ABS), a steroidogenesic disorder, was attributed to deleterious mutations of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), the obligatory electron donor for microsomal cytochrome P450-(CYP) catalyzed reactions. CYPOR contains both FAD and FMN cofactors. To probe the molecular basis of the observed deficiency, two ABS mutants, Y459H and V492E, were recombinantly expressed and purified. Wild-type (WT) CYPOR is green upon purification, due to the presence of a mixture of blue FMN semiquinone and yellow oxidized FAD. However, both Y459H and V492E were blue-gray, indicating a predominance of semiquinone. Flavin content showed that WT had a 50:50 ratio of FAD:FMN, whereas Y459H and V492E had 5:95 and 10:90, respectively. V492E retained 5% of cytochrome c reductase activity compared to WT while two phases were detected in the Y459H reaction: an initial phase with a rate ~20% of WT, and a prolonged second phase with a rate ~4% of WT. We hypothesized that Y459H was catalytically unstable, deteriorating to an inactive state after a small number of turnover events as a result of substitution of FMN at the FAD-binding site. Redox measurements of V492E showed similar potentials for EFMN-FMNH· and EFMNH·-FMNH2 couples (−48 mV and −280mV, respectively) compared to WT. However, transitions were observed at +160mV and −240mV, which are likely due to the presence FMN in the FAD-binding site. Redox measurements were hindered in Y459H by the instability of the protein. Diminished FAD-binding specificity, caused by both Y459H and V492E mutations, resulted in the CYPOR deficiency associated with ABS (Supported by NIH HL30050 to BSM).