Structure Function & Biosynthesis of Respiratory Enzymes

描述（由申请人提供）： 该提案将描述特定的酶如何控制活性电子的转移和分子氧的活化，同时最大限度地减少氧化损伤。这是细胞发育、健康和生存的核心。本研究计划包括酶反应机制、蛋白质结构与功能关系、蛋白质间相互作用、蛋白质翻译后修饰和生物电子远程转移机制的研究。在这些研究中将使用动力学、生物化学、光谱和结构研究以及定点诱变。该提案集中于蛋白质衍生的辅因子色氨酸甲萘醌（TTQ）的生物合成机制，以及新型二血红素酶MauG的结构和功能，所述MauG在甲胺脱氢酶（MADH）中的TTQ生物合成期间催化特定色氨酸残基的氧化和交联。MauG的底物是MADH的119-kDa前体蛋白，其具有单羟基化2 Trp 57并且没有交联。MauG催化底物的6电子氧化，导致2 Trp 57的第二次氧化、2 Trp 57和2 Trp 108的交联以及前两个反应的醌醇产物的氧化以形成氧化的TTQ。这些研究将描述一种新的氧活化生物学机制和使蛋白质中特定氨基酸残基易受氧化修饰的因素。这些结果将为开发将新的催化位点引入蛋白质和操纵酶结合血红素的功能的策略提供见解，并为如何减轻自然发生的蛋白质氧化损伤提供线索。正在进行的MADH-amicyanin-细胞色素c-551 i蛋白复合物中生物电子转移（ET）的机制研究将得到扩展，新的ET研究将用MauG启动。确定远程电子转移反应的机制将增强我们对呼吸和中间代谢在分子水平上的基本过程的理解。对生物ET反应控制机制的基本理解将深入了解有缺陷的蛋白质ET如何导致活性氧和自由基的产生，这两者都与许多疾病状态、氧化应激和衰老有关。公共卫生相关性：项目叙述：作为生物电子转移和氧代谢的副产物产生的活性氧物质和自由基对细胞组分造成非特异性氧化损伤，其引起线粒体肌病、许多疾病状态、氧化应激和衰老。然而，自由基和活性氧也是生物合成过程所必需的，并且在生物合成过程中得到了有效利用。该提案将阐明特定的酶如何控制活性电子的转移和分子氧的活化，同时最大限度地减少氧化损伤。

DESCRIPTION (provided by applicant): This proposal will describe how specific enzymes control the transfer of reactive electrons and the activation of molecular oxygen, while minimizing oxidative damage. This is central to cell development, health and survival. This research project includes studies of enzyme reaction mechanisms, protein structure-function relationships, protein-protein interactions, protein post- translational modification, and mechanisms of long range biological electron transfer. Kinetic, biochemical, spectroscopic and structural studies together with site-directed mutagenesis will be used in these studies. This proposal focuses on the mechanism of biosynthesis of the protein- derived cofactor, tryptophan tryptophylquinone (TTQ), and the structure and function of a novel di-heme enzyme MauG which catalyzes the oxygenation and cross-linking of specific tryptophan residues during TTQ biogenesis in methylamine dehydrogenase (MADH). The substrate for MauG is a 119-kDa precursor protein of MADH with mono-hydroxylated 2Trp57 and no cross- link. MauG catalyzes the 6-electron oxidation of the substrate that results in the second oxygenation of 2Trp57, cross-linking of 2Trp57 and 2Trp108, and oxidation of the quinol product of the first two reactions to form oxidized TTQ. These studies will describe a new biological mechanism for oxygen activation and factors that make specific amino acid residues in proteins susceptible to oxidative modification. The results will provide insight for development of strategies to introduce novel catalytic sites into proteins and manipulate the functions of enzyme-bound hemes, as well as provide clues as to how one might mitigate naturally occurring oxidative damage to proteins. Ongoing mechanistic studies of biological electron transfer (ET) in the MADH-amicyanin-cytochrome c-551i protein complex will be extended and new ET studies will be initiated with MauG. Defining the mechanisms of long range electron transfer reactions will enhance our understanding of the fundamental processes of respiration and intermediary metabolism at the molecular level. A fundamental understanding of the mechanisms of control of biological ET reactions will provide insight into how defective protein ET leads to production of reactive oxygen species and free radicals both of which are associated with many disease states, oxidative stress and aging. PUBLIC HEALTH RELEVANCE: Project Narrative: Reactive oxygen species and free radicals, which are produced as by-products of biological electron transfer and oxygen metabolism, cause non-specific oxidative damage to cell components that causes mitochondrial myopathies, many disease states, oxidative stress and aging. However, free radicals and reactive oxygen species are also required for, and used productively in biosynthetic processes. This proposal will elucidate how specific enzymes control the transfer of reactive electrons and the activation of molecular oxygen, while minimizing oxidative damage.