Control of Heme Enzyme Activity

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。特别令人感兴趣的是血红素蛋白的铁（III）态的化学反应性。将CcP的性质与两种氧运输/储存蛋白（哺乳动物metmy红蛋白（MetMb）和甘油三酯高铁血红蛋白（metGHb））的Fe（III）状态进行比较，以了解导致这两类血红素蛋白反应性差异的结构特征。要研究的铁（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，即铁（III）蛋白和H2O2之间形成被称为化合物I的氧化酶中间体的反应。最初的研究使用位点定向诱变来改变蛋白质结构，表明CcP中的两个氨基酸残基对化合物I的形成至关重要，远端组氨酸His-52和远端精氨酸（Arg-48）。Arg-48促进氧-氧键的裂解，而His-52促进过氧化物与血红素铁的结合。阐明过氧化物酶和氧运输/储存血红素蛋白中远端组氨酸特性的差异是本提案的具体目标之一。关于血红素蛋白Fe（III）状态的反应性的一个关键问题是小反应物和配体进入远端血红素口袋。通过研究小配体如氟化物、氰化物和一氧化氮与CcP、metMb、metGHb及其突变体中的铁的结合率，将探索活性位点通路。将确定配体的中性形式和带电形式之间的区别。空间约束、酸碱行为和血红素口袋的极性将通过位点定向诱变进行研究。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的理解。为蛋白质的合理设计提供依据。该提案包括将甲基甲氧基乙转化为高效过氧化物酶的计划，更重要的是，将CcP转化为一种通用的羟化酶，利用H2O2而不是O2。血红素蛋白在生物体中发挥着许多重要的功能，包括将氧气运输到其利用部位和保护生物体免受活性氧的侵害。（活性氧，如超氧化物、过氧化氢和羟基自由基被认为会加速衰老过程。）本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。两种具有不同功能的血红素蛋白将通过对其结构的系统操纵进行广泛的研究。观察改变后的蛋白质的生物反应性，就可以得出结构和功能方面的相关性。这些研究将为血红素蛋白的反应性和功能提供一个复杂的理解水平。这些研究也将为血红素蛋白的合理设计提供基础，以开展可能具有重要商业应用的工艺。＊＊＊

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. ***