Abstract
Seven of the 57 human cytochrome P450 (P450) enzymes are mitochondrial and carry out important reactions with steroids and vitamins A and D. These seven P450s utilize an electron transport chain that includes NADPH, NADPH-adrenodoxin reductase (AdR), and adrenodoxin (Adx) instead of the diflavin NADPH-P450 reductase (POR) used by the other P450s in the endoplasmic reticulum. Although numerous studies have been published involving mitochondrial P450 systems, the experimental conditions vary considerably. We compared human Adx and bovine Adx, a commonly used component, and found very similar catalytic activities in reactions catalyzed by human P450s 11B2, 27A1, and 27C1. Binding constants of 6-200 nM were estimated for Adx binding to these P450s using microscale thermophoresis. All P450 catalytic reactions were saturated at 10 μM Adx, and higher concentrations were not inhibitory up to at least 50 μM. Collectively these studies demonstrate the tight binding of Adx (both human and bovine) to AdR and to several mitochondrial P450s and provide guidance for optimization of Adx-dependent P450 reactions.