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 (Fe_BMb) and tuning its heme redox potentials (E°′) 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°′ 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°′ on NO reduction. Decreasing E°′ 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°′ 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°′ and fast electron transfer facilitated by high E°′. Only when E°′ is optimally tuned in Fe_BMb(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°′ in a similar range as NORs (between −59 and 200 mV) exhibit NOR reactivity. Thus, our work demonstrates efficient tuning of E°′ in various metalloproteins for their optimal functionality.

尽管NO还原酶（NORs）和血红素铜氧化酶（HCOs）之间具有高度的结构同源性，但控制其反应特异性的因素仍有待了解。使用基于肌红蛋白的NOR模型（Fe _B Mb）并通过操纵氢键结合至近端组氨酸配体并取代血红素b来调节其血红素氧化还原电势（E°'）以覆盖天然的NOR范围。对于单甲酰基（MF-）或二甲酰基（DF-）血红素，我们在本文中证明E°'是NOR和HCO之间反应性差异的关键。详细的电化学，动力学和振动光谱研究与密度泛函理论计算相结合，证明了血红素E°'对NO还原的强烈影响。E°'从+148降低到-130 mV会显着影响NOR模拟物的电子性能，分别导致NO缔合和血红素亚硝酰衰变速率分别提高180倍和633倍。我们的结果表明，NORs表现出微调的E°'，可最大程度地提高其酶促效率，并有助于实现相反因素之间的平衡：低E°'促进NO的快速结合和二亚硝酰基物质的分解，高E°'促进电子的快速转移。只有在铁中优化了E°'的情况下_B Mb（MF-血红素）具有NO结合，血红素-亚硝酰基衰变和电子转移的功能，使蛋白质实现了多个（> 35）转换，这是以前合成或基于酶的NOR模型无法实现的。这也解释了HCO中选择性交叉反应的生物能学中一个长期存在的问题。只有血红素E°'与NOR相似的HCO（在-59和200 mV之间）才显示NOR反应性。因此，我们的工作证明了各种金属蛋白中E°'的有效调节可实现其最佳功能。