Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.

血红蛋白介导的分子氧 (O ₂ ) 转运主要取决于血红素辅助因子的还原 (Fe ²⁺ ) 形式的稳定性。一些蛋白质突变稳定了氧化（Fe ³⁺）状态（高铁血红蛋白，Hb M），导致高铁血红蛋白血症，可致死率超过 30%。大多数对影响 Hb 氧化的因素的分析都是回顾性的，并且仅对血红素的内球突变（His58、His87）提供见解。在这里，我们报告了关于氧化还原状态的第一个全原子分子动力学模拟以及 Hb M 的α链 Hb 氧化和还原率的 Marcus 电子转移 (ET) 参数的计算。Hb 野生型 (WT) 和大多数研究的α除 Hb M Iwate (H87Y) 外，链变体保持珠蛋白结构。形成 Hb M 的突变体往往具有较低的氧化还原电位，因此稳定了氧化 (Fe ³⁺ ) 状态（特别是具有 K61E 突变的 Hb Miyagi 变体）。溶剂重组（λ _solv 73-96%）对重组自由能做出了重大贡献，而蛋白质重组（λ _prot）占 27-30%，除了 Miyagi 和 J-Buda 变体（λ _prot ∼4%）。分析变体之间的血红素-溶剂氢键相互作用提供了对 Lys61 残基在稳定 Fe ²⁺状态中的作用的见解。基于半经典 Marcus ET 理论的计算预测实验k_ET用于 Cyt b5-Hb 复合物，并提供有关 Hb 变体中 Hb M 的相对降低率的见解。因此，我们的方法为突变对结构、稳定性和 Hb 氧化还原率的影响提供了基本原理，并有可能识别导致高铁血红蛋白血症的突变。