Nonheme diiron enzymes catalyze a wide range of biologically essential redox transformations. Despite extensive study, how the surrounding protein matrix coordinates electron transfer, proton delivery, and solvent access and whether mechanistic insights derived in vitro reflect enzyme function in living systems remain incompletely understood. Here, we investigate the bacterial enzyme ScdA, a nonheme diiron nitrite reductase that converts nitrite to nitric oxide (NO), to define how first- and second-sphere interactions regulate catalysis. Using site-directed mutagenesis, steady-state kinetics, EPR spectroscopy, solvent kinetic isotope effect analysis, molecular dynamics simulations, and cell-based spin trapping, we identify distinct functional contributions of residues surrounding the diiron center. First-sphere ligands ensure cofactor assembly and redox integrity, whereas second-sphere residues modulate turnover by controlling hydrogen-bond networks and solvent accessibility near the catalytic core. Structural and kinetic analyses reveal a solvent-accessible pathway whose gating properties tune hydration dynamics and influence the rate-limiting step. Importantly, cell-based EPR detection of NO demonstrates that the same structural determinants governing catalytic efficiency in vitro also operate under cellular conditions. Together, these results establish controlled hydration as a general design principle in nonheme diiron enzymes.
Chen et al. (2026) studied this question.