Resonance Raman Spectroscopy and Density Functional Theory Reveal the Hemin Release Mechanism of Fish and Mammalian Hemoglobin

Salmonids are an incredibly valuable agricultural commodity, with large market growth expected over the next 10 years. Salmonids have a high feed conversion ratio and are rich in vital nutrients; however, their post-mortem tissues are subject to deterioration due in part to their labile hemoglobin (Hb). In this work, we aimed to understand the driving force for the pro-oxidative nature of salmonid (trout IV) Hb. We utilized resonance Raman spectroscopy (rR), electronic absorption spectroscopy (EA), and density functional theory (DFT) calculations to probe the distal and proximal heme pocket architectures of trout IV and bovine Hb. Using fluoride as a H-bond-sensitive ligand, we found that at low pH, trout IV ferric Hb is more likely to have a protonated distal His. The enhanced distal His protonation and mobility play a crucial role in hemin dissociation, explaining the oxidative capacity of salmonid Hb, which dwarfs that of mammalian Hb.鲑科鱼类（Salmonids）是极具经济价值的水产养殖商品，预计未来十年市场规模将实现大幅增长。这类水产不仅饲料转化率优异，还富含多种关键营养物质；然而，其死后组织易发生腐败变质，部分原因在于其体内的不稳定血红蛋白（Hb）。本研究旨在阐明鲑科鱼类（虹鳟IV型）血红蛋白促氧化特性的驱动机制。我们采用共振拉曼光谱法（resonance Raman spectroscopy, rR）、电子吸收光谱法（electronic absorption spectroscopy, EA）以及密度泛函理论（density functional theory, DFT）计算，对虹鳟IV型与牛血红蛋白的血红素远端及近端口袋结构进行了表征分析。以氟化物作为氢键敏感配体开展实验后发现，在低pH环境下，虹鳟IV型高铁血红蛋白更易出现远端组氨酸质子化现象。增强的远端组氨酸质子化作用及其流动性在血红素解离过程中发挥关键作用，这一机制正是鲑科鱼类血红蛋白氧化能力远超哺乳动物血红蛋白的原因。