Studies of the membrane-bound flavocytochrome MsrQ flavin mononucleotide (FMN)-binding site reveal an unexpected ubiquinone cofactor.

The methionine sulfoxide reductase PQ system (MsrPQ) is a newly identified type of bacterial methionine sulfoxide reductase (Msr) involved in the repair of periplasmic methionine residues that have been oxidized by hypochlorous acid. MsrP, which carries out the Msr activity, is a molybdoenzyme located in the periplasm, whereas MsrQ, an integral membrane-bound flavohemoprotein, specifically transfers electrons to MsrP to drive catalysis. MsrQ belongs to an important superfamily of heme-containing membrane-bound proteins, which includes the eukaryotic NADPH oxidases (NOX) and six-transmembrane epithelial antigen of the prostate (STEAP) ferric reductases. Like STEAP, and in addition to a b-type heme, MsrQ contains a flavin cofactor [flavin mononucleotide (FMN)], which mediates electron transfer from a cytosolic NADH oxidoreductase to the heme, and subsequently to MsrP. In this study, we characterized the FMN-binding site of MsrQ using an AlphaFold model, identifying R77 and R78 residues as potentially critical for FMN stabilization. The R77A and R78A mutations result in the complete loss of the FMN cofactor, showing that both residues are essential for FMN binding. Surprisingly, electron paramagnetic resonance (EPR) spectroscopy and biochemical analysis of the mutants revealed the presence of a ubiquinone (UQ) cofactor associated with MsrQ, independently of the binding of FMN. The mid-point redox potentials of the MsrQ heme and FMN cofactors, measured through redox titration and cyclic voltammetry experiments, contradict the previous assumption that UQ serves as the electron donor for MsrQ. Instead, our data suggest that UQ may function as an electron acceptor for the reduced form of MsrQ. We propose that UQ bound to MsrQ could act as a protective mechanism when MsrP substrate is limiting.