Control of Heme Enzyme Activity

血红素酶活性的控制

• 批准号: 9513047
• 负责人: James Erman
• 金额: $ 24万
• 依托单位: Northern Illinois University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9513047&HistoricalAwards=false
• 关键词: Control; Heme; Enzyme; Activity

9513047 Erman The long-term goal of this research is to elucidate the structural features which control heme protein reactivity and determine heme protein function. The research will compare proteins from two different functional classes: (1) the heme enzymes and (2) the oxygen transport/storage proteins. Of particular interest is the chemical reactivity of the Fe(III) state of heme proteins. The properties of CcP will be compared with those of the Fe(III) state of two oxygen transport/storage proteins, mammalian metmyoglobin (MetMb) and Glycera dibranchiata methemoglobin (metGHb) in an effort to understand the structural features which are responsible for the reactivity differences in these two classes of heme proteins. The first aspect of Fe(III) heme protein reactivity to be investigated is the reaction which characterizes the peroxidases, the reaction between the Fe(III) protein and H2O2 to form an oxidized enzyme intermediate called Compound I. Initial studies, using site-directed mutagenesis to alter protein structure, indicate that two amino acid residues in CcP are critical in the formation of Compound I, the distal histidine His-52 and a distal arginine (Arg-48). Arg-48 facilitates oxygen-oxygen bond cleavage while His-52 promotes peroxide binding to the heme iron. Elucidation of the differences in the properties of the distal histidine in the peroxidases and the oxygen transport/storage heme proteins is one of the specific objectives of this proposal. A key question concerning the reactivity of the Fe(III) state of heme proteins is access of small reactants and ligands to the distal heme pocket. Active-site access will be probed by investigating the rates of binding of small ligands such as fluoride, cyanide, and nitric oxide to the iron in CcP, metMb, metGHb and their mutants. Discrimination between the neutral and charged forms of the ligands will be determined. Steric constraints, acid-base behavior, and the polarity of the heme pocket will be investigated through site-directed mutagenesis. The studies outlined in this proposal will provide a more sophisticated level of understanding of heme protein reactivity and function. It will provide a basis for rational protein design. The proposal includes plans to convert metGHb into an efficient peroxidase and more importantly, to convert CcP into a versatile hydroxylase, utilizing H2O2 rather than O2. %%% Heme proteins perform many important functions in living organisms including the transport of oxygen to its site of utilization and the protection of the organism from reactive oxygen species. (Reactive oxygen species such as superoxide, hydrogen peroxide, and the hydroxyl radical are thought to accelerate the aging process.) The goal of the research proposed in this project is to determine how the structure of various heme proteins govern their biological function. Two heme proteins, with different functions, will be extensively investigated by systematic manipulation of their structures. Observation of the biological reactivity of the altered proteins will allow correlations to be made concerning structure and function. The studies will provide a sophisticated level of understanding of heme protein reactivity and function. These studies will also provide a basis for the rational design of heme proteins to carry out processes which may have important commercial applications. ***

9513047 Erman本研究的长期目标是阐明控制血红素蛋白反应性和确定血红素蛋白功能的结构特征。该研究将比较两种不同功能类别的蛋白质：（1）血红素酶和（2）氧运输/储存蛋白质。特别感兴趣的是血红素蛋白的Fe（III）状态的化学反应性。CcP的性质将进行比较与Fe（III）的状态的两个氧运输/储存蛋白，哺乳动物高铁肌红蛋白（MetMb）和二鳃甘油高铁血红蛋白（metGHb）在努力了解的结构特征，这是负责这两类血红素蛋白的反应性差异。研究Fe（III）血红素蛋白反应性的第一个方面是表征过氧化物酶的反应，即Fe（III）蛋白和H2 O2之间形成称为化合物I的氧化酶中间体的反应。使用定点诱变改变蛋白质结构的初步研究表明，CcP中的两个氨基酸残基在化合物I的形成中是关键的，即远端组氨酸His-52和远端精氨酸（Arg-48）。Arg-48促进氧-氧键断裂，而His-52促进过氧化物与血红素铁结合。 阐明远端组氨酸在过氧化物酶和氧转运/储存血红素蛋白的性质的差异是本提案的具体目标之一。 一个关键的问题，关于血红素蛋白的Fe（III）状态的反应性是访问小的反应物和配体的远端血红素口袋。活性位点的访问将通过调查的结合率的小配体，如氟化物，氰化物，一氧化氮的铁在CcP，metMb，metGHb和它们的突变体进行探测。将确定配体的中性和带电形式之间的区别。将通过定点突变研究空间限制、酸碱行为和血红素口袋的极性。本提案中概述的研究将提供对血红素蛋白反应性和功能的更复杂水平的理解。为蛋白质的合理设计提供了依据。该提案包括计划将metGHb转化为有效的过氧化物酶，更重要的是，利用H2 O2而不是O2将CcP转化为多功能羟化酶。 %血红素蛋白在生物体中执行许多重要功能，包括将氧运输到其利用部位和保护生物体免受活性氧物种的影响。（活性氧物质，如超氧化物，过氧化氢和羟基自由基被认为加速老化过程。该项目提出的研究目标是确定各种血红素蛋白的结构如何控制其生物学功能。两个血红素蛋白，具有不同的功能，将被广泛的研究，通过系统的操纵他们的结构。对改变的蛋白质的生物反应性的观察将允许进行关于结构和功能的相关性。这些研究将提供一个复杂的水平血红素蛋白的反应性和功能的理解。这些研究也将为血红素蛋白的合理设计提供基础，以进行可能具有重要商业应用的过程。 ***