Measurement and Control of Crossed Potentials in a Flavoprotein

Flavoproteins constitute 1–3% of prokaryotic and eukaryotic proteins, functioning as electron transfer agents, catalysts, and sensing or regulatory modules. Their versatility as redox-active proteins stems from the tunability of the flavin cofactor’s one- and two-electron reduction potentials via interactions with the protein scaffold. Although several mechanisms have been proposed to explain how the flavin-binding pocket modulates redox thermodynamics, a holistic model enabling interpretation and prediction remains to be established. In this study, we investigate how the flavin N5 environment influences the redox properties of the flavin mononucleotide cofactor in the “improved” light-oxygen-voltage (iLOV) sensing protein using site-directed mutagenesis, redox titrations, and hybrid quantum mechanical molecular mechanical (QM/MM) methods combined with classical alchemical free energy simulations. Mutating the residue Q 103, which interacts with the flavin N5 and O4 atoms in the X-ray crystallographic structure, exerts a modest