CYTOCHROME B5-HEMOGLOBIN REDOX COUPLE

细胞色素 B5-血红蛋白氧化还原对

• 批准号: 3276161
• 负责人: ARTHUR G MAUK
• 金额: $ 5.25万
• 依托单位: UNIVERSITY OF BRITISH COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-09-01 至 1986-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276161
• 关键词: NAD(H) analog; NADPH cytochrome c2 reductase; blood proteins; chemical models; crosslink; cytochrome b; cytochrome b5 reductase; cytochrome c; cytochrome oxidase; electrolyte balance; electron transport; erythrocytes; hemopexin; mental retardation; metalloproteins; methemoglobin; myoglobin; oxidation reduction reaction; physical chemical interaction

In the erythrocyte, methemoglobin (metHb) is reduced to ferroHb primarily by reaction with ferrocytochrome b5 which mediates the transfer of electrons from NADH-dependent metHb reductase. This recycling of the non-functional metHb is essential to prevent accumulation of the oxidized protein during the 120 day life span of the red cell since 0.5-3% of circulating Hb is converted to metHb each day. Deficiencies in this reductase activity result in methemoglobinemia. Combined erythrocyte and brain deficiencies (related enzymes occur in most tissues) produce methemoglobinemia, mental retardation and early death. As the 3-dimensional structures of cytochrome b5 and metHb are known to high resolution, we are developing a detailed model for the electron transfer reaction between these two proteins that should define an unexplored area of Hb physiology and provide a working model for electron transfer reactions between other metalloproteins (e.g., the reaction of cytochrome c with cytochrome oxidase). Parallel studies are also outlined for the simpler but closely related reaction between the well-characterized proteins cytochrome b5 and cytochrome c. Features of this reaction mechanism that will be investigated include (1) identification of amino acid residues on the two proteins that are responsible for protein-protein recognition through use of chemical modification and crosslinking techniques, (2) determination of stability of the cytochrome b5-metHb complex at physiological pH and ionic strength by fluorescence quenching titrations, (3) estimation of the distance between redox centers of the two proteins by energy transfer measurements and determination of the functional dependence of the rate of electron transfer on this distance, and (4) preparation and structural and functional characterization of crosslinked metHb-cytochrome b5 complexes. In addition, we will explore the possibility that the heme exchange reaction between metHb and apocytochrome b5 is a reasonable model for the transfer of heme from metHb to hemopexin, a heme-scavenging protein from plasma known to have surface properties and heme-binding characterization very similar to these of cytochrome b5.

在红细胞中，高铁血红蛋白（metHb）主要被还原为铁血红蛋白（ferroHb）。 通过与铁细胞色素b5反应， 电子来自NADH依赖的metHb还原酶。 这种循环利用 非功能性高铁血红蛋白是必不可少的，以防止积累的氧化 在红细胞的120天寿命期间， 每天循环的Hb转化为metHb。 防御在这 还原酶活性导致高铁血红蛋白血症。 结合红细胞和 脑缺陷（相关酶存在于大多数组织中）产生 高铁血红蛋白血症、智力迟钝和早逝。 为 3-已知细胞色素b5和metHb的三维结构具有高的 分辨率，我们正在开发一个详细的电子转移模型， 这两种蛋白质之间的反应应该定义一个未探索的区域 并提供电子转移的工作模型 其它金属蛋白之间的反应（例如，细胞色素c反应 用细胞色素氧化酶）。 还概述了对以下方面的平行研究： 简单但密切相关的反应之间的良好表征 蛋白质细胞色素b5和细胞色素c。 该反应的特点 将研究的机制包括（1）鉴定氨基 负责蛋白质-蛋白质的两种蛋白质上的酸残基 通过使用化学改性和交联的识别 技术，（2）测定细胞色素b5-metHb的稳定性 复合物在生理pH和离子强度通过荧光猝灭 滴定，（3）估算两个氧化还原中心之间的距离 蛋白质的能量转移测量和测定 电子转移速率对该距离的函数依赖性， 和（4）制备和结构和功能表征 交联的metHb-细胞色素b5复合物。 此外，我们还将探讨 高铁血红蛋白与亚铁血红素之间的血红素交换反应的可能性 脱辅基细胞色素b5是从高铁血红蛋白转移血红素的合理模型 血红素结合蛋白，一种来自血浆的血红素清除蛋白， 性质和血红素结合特征非常相似， 细胞色素b5。