Insights into the Direct Photoelectron Transfer Mechanism in Cofactor-free Redox Carbon Dots and Cytochrome <i>c</i> Nitrite Reductase Biohybrids Responsible for Ammonia Synthesis

Highly efficient NH₃ production has been reported to be achieved by photosensitizing <i>Shewanella oneidensis</i> MR-1 using carbon dots (CDs). During this process, cytochrome <i>c</i> nitrite reductase (NrfA) is regarded as the rate-limiting enzyme. However, the precise electron transfer mechanism between CDs and NrfA remains unclear. Herein, a hybrid photosynthetic system composed of NrfA and redox CDs (NrfA/R-CDs) was constructed, achieving a maximum NH₃ production rate of 12.5 ± 1.1 μmol (NH₃)·mg^-1 (NrfA)·h^-1. R-CDs with aromatic ketone groups could store photoinduced electrons, enhancing carrier separation efficiency. These stored photoelectrons were capable of being directly transferred to NrfA without the need of cofactors. Even under dark conditions, direct electron transfer occurred from the stored photoelectrons in R-CDs to NrfA, providing an indirect illumination approach to reduce phototoxicity to NrfA. Molecular docking and dynamics simulations demonstrated the formation of a stable complex between the heme 2 region of NrfA and R-CDs. The short distance (10 Å) and π-electronic interactions between R-CDs and heme 2 facilitated the direct electronic transfer process. This study provides a comprehensive understanding of the interfacial photoelectron transfer mechanism and guidelines for constructing nanomaterials with photoelectron storage and release properties.