Reeder Brandon J, Grey Marie, Silaghi-Dumitrescu Radu-Lucian, Svistunenko Dimitri A, Bülow Leif, Cooper Chris E, Wilson Michael T
Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK.
J Biol Chem. 2008 Nov 7;283(45):30780-7. doi: 10.1074/jbc.M804709200. Epub 2008 Aug 26.
Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.
诸如肌红蛋白和血红蛋白之类的呼吸蛋白在氧化条件下可形成高铁血红素铁和基于蛋白质的自由基。高铁肌红蛋白可通过外源还原剂的电子供体经由涉及两条不同途径的机制安全地恢复到三价氧化态。除了电子供体与高铁血红素边缘之间的直接转移外，还有第二条途径，即通过酪氨酸残基（酪氨酸103，抹香鲸肌红蛋白）进行“蛋白质内”电子转移。在这里，我们表明人类血红蛋白的异源亚基α链和β链对外源还原剂还原高铁血红素显示出显著不同的动力学。通过使用选定的血红蛋白突变体，我们表明α链拥有两条类似于肌红蛋白的电子转移途径。此外，酪氨酸42被证明是高亲和力蛋白质内电子转移途径的关键组成部分。我们还表明，缺乏同源酪氨酸的血红蛋白β链不具备这种蛋白质内电子转移途径。然而，通过将特定的苯丙氨酸残基突变为酪氨酸，可以在蛋白质中构建这样一条途径。高亲和力的蛋白质内电子转移途径，无论是天然的还是构建的，都能增强还原剂去除高铁血红素的动力学，特别是在低还原剂浓度下。高铁血红素铁被认为是基于血红蛋白的血液替代品氧化毒性的主要原因。因此，如我们在此所示，构建具有更高高铁血红素去除率的血红蛋白可能会产生更适合体内氧气输送的分子。
