Bound Flavin–Cytochrome Model of Extracellular Electron Transfer in <i>Shewanella oneidensis</i>: Analysis by Free Energy Molecular Dynamics Simulations

Flavins are known to enhance extracellular electron transfer (EET) in <i>Shewanella oneidensis</i> MR-1 bacteria, which reduce electron acceptors through outer-membrane (OM) cytochromes <i>c</i>. Free-shuttle and bound-redox cofactor mechanisms were proposed to explain this enhancement, but recent electrochemical reports favor a flavin-bound model, proposing two one-electron reductions of flavin, namely, oxidized (Ox) to semiquinone (Sq) and semiquinone to hydroquinone (Hq), at anodic and cathodic conditions, respectively. In this work, to provide a mechanistic understanding of riboflavin (RF) binding at the multiheme OM cytochrome OmcA, we explored binding configurations at hemes 2, 5, 7, and 10. Subsequently, on the basis of molecular dynamics (MD) simulations, binding free energies and redox potential shifts upon RF binding for the Ox/Sq and Sq/Hq reductions were analyzed. Our results demonstrated an upshift in the Ox/Sq and a downshift in the Sq/Hq redox potentials, consistent with a bound RF–OmcA model. Furthermore, binding free energy MD simulations indicated an RF binding preference at heme 7. MD simulations of the OmcA–MtrC complex interfacing at hemes 5 revealed a small interprotein redox potential difference with an electron transfer rate of 10⁷–10⁸/s