Activation of Coq6p, a FAD Monooxygenase Involved in Coenzyme Q Biosynthesis by Adrenodoxin Reductase/Ferredoxin

Adrenodoxin reductase (AdxR) plays a pivotal role in electron transfer, shuttling electrons between NADPH and iron/sulfur adrenodoxin proteins in mitochondria. This electron transport system is essential for P450 enzymes involved in various endogenous biomolecules biosynthesis. Here, we present an in‐depth examination of the kinetics governing the reduction of human AdxR by NADH or NADPH. Our results highlight the efficiency of human AdxR when utilizing NADPH as a flavin reducing agent. Nevertheless, akin to related flavoenzymes such as cytochrome P450 reductase, we observe that low NADPH concentrations hinder flavin reduction due to intricate equilibrium reactions between the enzyme and its substrate/product. Remarkably, the presence of MgCl2 suppresses this complex kinetic behavior by loosening NADPH binding to oxidized AdxR, effectively transforming AdxR into a classical Michaelis‐Menten enzyme. We propose that the addition of MgCl2 may be adapted for studying the reductive half‐reactions of other flavoenzymes with NADPH. Furthermore, in vitro experiments provide evidence that the reduction of the yeast flavin monooxygenase Coq6p relies on an electron transfer chain comprising NADPH‐AdxR‐Yah1p‐Coq6p, where Yah1p functions as a ferredoxin. This discovery explains the previous in vivo observation that Yah1p and the AdxR homolog, Arh1p, are required for the biosynthesis of coenzyme Q in yeast.