Structure Function & Biosynthesis of Respiratory Enzymes

该提案描述了特定的酶如何控制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。本项目包括酶反应机理、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理的研究。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。本论文主要研究了蛋白质衍生的辅因子色氨酸甲萘醌（TTQ）的生物合成机制，以及一种新型的二血红素酶MauG的结构和功能，MauG在甲胺脱氢酶（MADH）中催化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MADH的119-kDa前体蛋白，具有单羟基化Trp 57并且没有交联。MauG催化底物的6电子氧化，导致Trp 57的第二次氧化、Trp 57和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，其结果将为开发将新催化位点引入蛋白质和操纵酶结合血红素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，ET研究将用MauG启动。确定远距离ET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。 相关性（参见说明）： 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损害引起许多疾病状态、氧化应激和衰老的细胞组分。然而，自由基和活性氧也被有效地用于生物合成过程。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，同时最大限度地减少氧化损伤。

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.