Structure Function & Biosynthesis of Respiratory Enzymes

这项建议描述了特定的酶如何控制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。该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远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。本研究的重点是蛋白质衍生辅因子色氨酸色酚(TTQ)的生物合成机制，以及在甲胺脱氢酶(MADH)TTQ生物合成过程中催化特定色氨酸残基氧化和交联的新型双血红素酶MAUG的结构和功能。MAUG的底物是一种119 kDa的MADH前体蛋白，含有单羟基化的Trp57，没有交联键。MAUG催化底物的6电子氧化，导致Trp57的第二次氧化，Trp57和Trp108的交联，以及前两个反应的喹酚产物氧化形成氧化的TTQ。这些研究将描述氧激活的新的生物学机制以及使蛋白质中的特定氨基酸残基易于氧化修饰的因素。这些结果将为在蛋白质中引入新的催化位点和操纵酶结合的血红素的功能的策略的开发提供洞察力，并为如何减轻蛋白质自然发生的氧化损伤提供线索。正在进行的MADH-氨基蓝蛋白-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，并将与MAUG一起启动ET研究。明确远程ET反应的机制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。 相关性(请参阅说明)： 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会破坏细胞成分，导致许多疾病状态、氧化应激和衰老。然而，在生物合成过程中，自由基和活性氧物种也被大量使用。这些研究描述了特定的酶如何控制电子的转移和激活氧气，同时将氧化损伤降至最低。

This proposal describes how specific enzymes control the transfer of electrons and activation of molecular oxygen, while minimizing oxidative damage. This is central to cell development, health and survival. This 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 ofthe 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 Trp57 and no cross-link. MauG catalyzes the 6-electron oxidation of the substrate that results in the second oxygenation of Trp57, cross-linking of Trp57 and Trp108, 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-551 i protein complex will be extended and ET studies will be initiated with MauG. Defining mechanisms of long range ET 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. RELEVANCE (See instructions): Reactive oxygen species and free radicals produced as by-products of biological electron transfer and oxygen metabolism damage cell components that cause many disease states, oxidative stress and aging. However, free radicals and reactive oxygen species are also used productively in biosynthetic processes. These studies describe how specific enzymes control the transfer of electrons and activate oxygen, while minimizing oxidative damage.