Structure Function & Biosynthesis of Respiratory Enzymes

描述(由申请人提供)： 这项提案将描述特定的酶如何控制反应电子的转移和分子氧的激活，同时将氧化损伤降至最低。这对细胞发育、健康和生存至关重要。本研究项目包括酶反应机制、蛋白质结构-功能关系、蛋白质-蛋白质相互作用、蛋白质翻译后修饰和长程生物电子转移机制的研究。动力学、生化、光谱和结构研究以及定点突变将用于这些研究。这项建议集中在蛋白质衍生的辅因子色氨酸色酚(TTQ)的生物合成机制，以及一种新的双血红素酶MAUG的结构和功能，该酶催化甲胺脱氢酶(MADH)中TTQ生物合成过程中特定色氨酸残基的氧化和交联。MAUG的底物是一个119 kDa的MADH前体蛋白，2Trp57单羟基，没有交联键。MAUG催化底物的6电子氧化，导致2Trp57的第二次氧化，2Trp57和2Trp108的交联，以及前两个反应的喹酚产物氧化生成氧化的TTQ。这些研究将描述一种新的氧激活的生物学机制，以及使蛋白质中特定氨基酸残基易于氧化修饰的因素。这些结果将为开发将新的催化位点引入蛋白质并操纵酶结合的血红素的功能的策略提供洞察力，并为如何减轻对蛋白质的自然氧化损伤提供线索。正在进行的MADH-氨基青素-细胞色素c-551i蛋白复合体中生物电子转移(ET)的机制研究将得到扩展，新的ET研究将与MAUG一起启动。明确长程电子转移反应的机理将在分子水平上加深我们对呼吸和中间代谢的基本过程的理解。对生物ET反应控制机制的基本了解将有助于深入了解蛋白质ET缺陷如何导致活性氧物种和自由基的产生，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品产生的活性氧物种和自由基，对细胞成分造成非特异性氧化损伤，导致线粒体肌病、许多疾病状态、氧化应激和衰老。然而，自由基和活性氧物种也是生物合成过程所必需的，并在生物合成过程中有效地使用。这项建议将阐明特定的酶如何控制反应电子的转移和分子氧的激活，同时将氧化损伤降至最低。

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.