QM/MM Unlocks The Catalytic Mechanism of YtfE: The Structural Reorganization of The Active Site Enables Nitrite‐to‐NO Conversion

Abstract Escherichia coli ( E. coli ) repair of iron center protein (also known as YtfE) is a nonheme diiron protein that plays a pivotal role in bacterial responses to nitrosative stress, particularly in nitrite reduction and nitric oxide metabolism. Although previous studies have explored its potential as a nitrite reductase, the detailed catalytic mechanism, especially the role of its diiron center in nitrite reduction, remains poorly understood. In this study, we employed multiscale computational approaches, including quantum mechanics/molecular mechanics (QM/MM), quantum mechanics/molecular mechanics metadynamics (QM/MM MD), and quantum mechanics (QM) calculations, to elucidate the molecular mechanism of nitrite reduction by YtfE. Our findings reveal that structural reorganization of the active site, particularly the alternation of the hydrogen bonding network and coordination switch of the HONO intermediate, is critical for modulating the nitrite reduction mechanism. This work provides significant insights into the catalytic mechanism of YtfE, offering a theoretical foundation for understanding its role in bacterial stress responses and potential applications in synthetic biology and environmental remediation.