Cell-Surface Inter-Cytochrome Electron Transfer Limits Biofilm Electron Conduction Kinetics in Shewanella oneidensis

The electrical conductivity of biofilms plays a critical role in advancing bioelectronics for energy and environmental applications, yet the underlying mechanisms remain poorly understood. Previous studies proposed interheme electron transfer between hemes 5 and 10 in the outer-membrane deca-heme cytochrome (OMC) MtrC as the rate-limiting step in the biofilm electron conduction of Shewanella oneidensis MR-1. However, the strong interheme electron coupling in MtrC suggests that interprotein interactions may represent the primary barrier to biofilm electron conduction. Here, we investigated the biofilm electron conduction mechanism with a focus on interprotein electron transfer in S. oneidensis MR-1. Conductive currents and their temperature dependence were measured for estimating the thermal activation energy (E_a) by using indium tin-doped oxide (ITO) interdigitated electrodes in wild-type and mutant biofilms. While deletion of periplasmic cytochromes had a negligible impact on E_a, the deletion of OmcA or MtrC increased E_a 3-fold, revealing that interprotein interactions, particularly between OmcA and MtrC, dominate biofilm electron transfer over clonal OMC interactions. Furthermore, suppressing outer-membrane fluidity dramatically increased E_a, while interheme exciton coupling negligibly changed in the OMCs, confirming the critical role of protein diffusion and collision on the outer membrane. Flavin binding to OmcA or MtrC reduced conduction currents attributable to heme centers but enhanced those assignable to noncovalently bound flavins, suggesting that flavin occupancy blocks hemes 2 and 7, which serve as key interprotein electron transfer sites. These findings provide a mechanistic foundation for engineering highly conductive biofilms through targeted protein interface optimization, offering new avenues for the development of bioelectronic technologies.