Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase

Despite high structural homology between NO reductases (NORs) and heme-copper oxidases (HCOs), factors governing their reaction specificity remain to be understood. Using a myoglobin-based model of NOR (FeBMb) and tuning its heme redox potentials (E degrees') to cover the native NOR range, through manipulating hydrogen bonding to the proximal histidine ligand and replacing heme b with monoformyl (MF-) or diformyl (DF-) hemes, we herein demonstrate that the E degrees' holds the key to reactivity differences between NOR and HCO. Detailed electrochemical, kinetic, and vibrational spectroscopic studies, in tandem with density functional theory calculations, demonstrate a strong influence of heme E degrees' on NO reduction. Decreasing E degrees' from + 148 to -130 mV significantly impacts electronic properties of the NOR mimics, resulting in 180- and 633-fold enhancements in NO association and heme-nitrosyl decay rates, respectively. Our results indicate that NORs exhibit finely tuned E degrees' that maximizes their enzymatic efficiency and helps achieve a balance between opposite factors: fast NO binding and decay of dinitrosyl species facilitated by low E degrees' and fast electron transfer facilitated by high E degrees'. Only when E degrees' is optimally tuned in FeBMb(MF-heme) for NO binding, heme-nitrosyl decay, and electron transfer does the protein achieve multiple (>35) turnovers, previously not achieved by synthetic or enzyme-based NOR models. This also explains a long-standing question in bioenergetics of selective cross-reactivity in HCOs. Only HCOs with heme E degrees' in a similar range as NORs (between -59 and 200 mV) exhibit NOR reactivity. Thus, our work demonstrates efficient tuning of E degrees' in various metalloproteins for their optimal functionality.