BACTERIAL PROTEINS CONTAINING NOVEL IRON SITES

含有新铁位点的细菌蛋白

• 批准号: 2180301
• 负责人: DONALD M. KURTZ
• 金额: $ 13.96万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1996-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180301
• 关键词: Desulfovibrio; Raman spectrometry; X ray crystallography; bacterial proteins; chemical binding; electron spin resonance spectroscopy; hemerythrin; iron compounds; metalloproteins; molecular cloning; molecular site; nuclear magnetic resonance spectroscopy; open reading frames; oxidation reduction reaction; oxygen transport; peroxidases; protein structure function; site directed mutagenesis

Two proteins, rubrerythrin (Rr) and hemerythrin (Hr), are proposed for study. Both of these proteins contain non-heme diiron sites with the latter containing in addition a rubredoxin (Rd)-type FeS4 site. The former protein appears to be involved in O2 activation or scavenging, whereas the latter is known to bind O2 reversibly. Structural and functional characterizations of these iron sites and their interactions with the surrounding polypeptides is the overall focus of the proposed research. The study of these two proteins together is designed to foster a synergism in understanding the properties of their similar, yet distinct diiron sites. Rr was originally isolated from Desulfovibrio vulgaris, a strictly anaerobic, sulfate-reducing bacterium. Its biological function and the structures of its metal sites have not yet been established. However, the gene sequence for Rr, together with spectroscopic and biochemical results allow several hypotheses to be put forward. These hypotheses include the following: i) the diiron site of Rr reacts with peroxides to form a high-valent species capable of oxidizing certain electron donors, i.e., Rr can function as a peroxidase. ii) two conserved amino acid sequences in Rr furnish ligands to the diiron site. iii) the Rd-type site functions as an electron donor/acceptor for the diiron site in Rr. iv) the Rr gene is part of an operon, at least one of whose proteins functions as electron donor/acceptor for Rr. Experimental tests of these hypotheses include sequencing of Rr genes from at least two other species, sequencing and overexpression of regions flanking the D. vulgaris Rr gene, characterization of a reconstituted Rr which is enriched in diiron sites, and site-directed mutagenesis of Rr that has been cloned and overexpressed in E. coli. Peroxidase activities of the reconstituted and recombinant, mutated Hrs will show whether this activity requires diiron sites. Site-directed mutagenesis of recombinant Hr is proposed in order to probe in detail the requirements for reversible O2 binding. Histidine ligands will be replaced with carboxylates, and side-chains capable of hydrogen bonding to the iron ligands will be altered. These changes should have predictable effects on O2 affinity. Exposure of the O2 binding site to solvent by substitution of less bulky surrounding side chains could either raise or lower O2 affinity depending on the relative effects of solvent exposure on O2 dissociation vs association rates. These predictions will be tested by measuring the O2 affinities and O2 dissociation rates of the mutated Hrs. Attempts will also be made to change the diiron site of Hr into one that activates O2 by removal of one of the bridging carboxylates and replacement of some of the histidine ligands with carboxylates. Hrs from almost all species do not bind O2 in a cooperative manner, but Hr from the brachiopod Lingula reevii is an exception. The discovery that two non-identical subunits are present in approximately equal amounts in this Hr raises the possibility that the cooperativity is due to an alpha4beta4 subunit composition. This hypothesis will be tested by separately cloning the alpha and beta subunits and measuring the O2 binding curves of the two types of subunits. These studies are aimed at a clearer understanding of O2 binding and O2 activation in biological systems.

提出了两种蛋白质，rubreryThrin（RR）和Hymerythrin（HR） 学习。 这两种蛋白质都包含非血红素二氮位点 后者还含有Rubredoxin（RD）-Type Fes4位点。 这 以前的蛋白质似乎参与了O2激活或清除 而后者已知可以可逆地绑定O2。 结构和 这些铁位点及其相互作用的功能表征 与周围的多肽一起是提议的总体重点 研究。 对这两种蛋白质的研究旨在促进 理解其相似特性的协同作用 独特的二龙网站。 RR最初是从Desulfovibrio中分离出来的 寻常，严格的厌氧，硫酸盐还原细菌。 它是 生物功能及其金属位点的结构尚未 已建立。 但是，RR的基因序列，以及 光谱和生化结果允许提出几个假设 向前。 这些假设包括以下内容：i） RR与过氧化物反应，形成一种能够 氧化某些电子供体，即RR可以充当过氧化物酶。 ii）RR中的两个保守的氨基酸序列将配体提供给 二龙网站。 iii）RD型站点充当电子 RR中的Diiron网站的捐助者/受体。 iv）RR基因是一个 操纵子，至少一种蛋白质作为电子作用 RR的捐助者/受体。 这些假设的实验测试包括 来自至少两个其他物种的RR基因的测序，即测序和 D. vulgaris rr基因侧面的区域的过表达， 重构RR的表征，该RR富含二龙网站， 以及已克隆的RR的位置定向诱变， 大肠杆菌过表达。 重构的过氧化物酶活性和 重组，突变的HRS将显示此活动是否需要二龙 站点。 提出重组HR的位置定向诱变按顺序 详细探讨可逆O2绑定的要求。 组氨酸 配体将被羧酸盐和侧链代替 将氢键合在铁配体上将改变。 这些变化 应该对O2亲和力产生可预测的影响。 O2的暴露 通过替换较小的周围侧来结合溶剂的结合位点 链可能会提高或降低O2亲和力，具体取决于亲戚 溶剂暴露对O2解离与关联率的影响。 这些预测将通过测量O2亲和力和O2进行测试 突变的HRS解离速率。 也将尝试 将人力资源的二铁部位更改为一个通过去除一个来激活O2的位置 桥接的羧酸盐和一些组氨酸的替代 配体配羧酸盐。 来自几乎所有物种的HR不结合O2 以合作的方式，但是腕足动物lingula reevii的人力资源是一个 例外。 发现两个非相同亚基存在的发现 在此人力资源中，大约相等的数量增加了 协作性是由于α4Beta4亚基组成。 这 假设将通过单独克隆α和beta进行检验 亚基并测量两种类型的O2结合曲线 亚基。 这些研究的目的是对O2有更清晰的了解 生物系统中的结合和O2激活。