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, which interacts with the flavin N5 and O4 atoms in the X-ray crystallographic structure, exerts a modest <35 mV effect on the overall two-electron reduction potential, but significantly alters the potential separation of the two one-electron couples (potential crossing) by up to 168 mV. QM/MM and free energy calculations reveal that water penetration into the flavin-binding pocket near N5 and O4 largely explains the trend in reduction potentials among the mutants. The results suggest a molecular mechanism of flavin tuning in which hydrogen bonding to the neutral semiquinone, either directly by the side-chain or a protein-penetrating water, contributes significantly to the potential crossing. These findings establish quantitative experimental benchmarks for theoretical models and advance a molecular mechanism for redox tuning in flavoproteins.