Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

生物大分子之间的电子转移是许多生物过程的基础。它通常发生在一个瞬时复合体中，其中参与氧化还原中心和中间蛋白质的最佳配置是促进和调节中心之间的电子流动。这类络合物很难从结构上进行研究，因为它们本质上是不稳定的，通常不能结晶。脱氮副球藻的甲胺脱氢酶(MADH)、氨基花青素和细胞色素C551I形成了最具特征的生理电子传递复合体之一，也是唯一具有高分辨结构的三种可溶性氧化还原蛋白的复合体。MADH是一个大小为124 kDa的2.2异二聚体，含有不寻常的氧化还原辅因子色氨酸色氨酸(TTQ)。氨蓝蛋白是一种大小为12.5 kDa的蓝铜蛋白，能与MADH特异结合。细胞色素C551I是一种17.5 kDa的蛋白质，它可以接受氨蓝蛋白的电子，并通过另一种细胞色素将电子转移到末端的氧化酶上。粪产碱菌的芳香胺脱氢酶(AADH)在大小和四级结构上与MADH相似，并含有TTQ作为氧化还原辅因子。然而，其特异的电子受体是天青素，MADH和AADH都不能有效地还原替代受体蛋白天青素或氨基蓝。该建议的目的是(1)在原子分辨率(1.或更低)下提纯在pH 5.5和8.0下氧化和还原的氨基花青素的结构，(2)开展MADH/氨花青素和MADH/氨花青素/c551I络合物的额外氧化还原和pH变异体和催化中间体的结构研究，(3)表征这些络合物中氨花青素和MADH的突变体的结构特征，(4)进一步分析来自甲基鞭毛杆菌KT和甲基乳杆菌AM1的MADH的氧化还原和阳离子结合状态，对MADH和AADH酶系统的研究将有助于确定对电子转移具有重要意义的结构特征，包括参与伙伴识别、电子转移控制和确定电子在络合物中流动路径的结构特征。此外，对TTQ辅因子底物氧化机制的结构研究将有助于阐明该辅因子在体内如何发挥作用，以及它与其他更常见的氧化还原辅因子有何不同。TTQ是不同寻常的，因为它是由基因组DNA编码的两个氨基酸侧链直接融合而成，而不是通过单独的生物合成途径获得的。MADH和AADH系统非常适合在分子水平上提供对这些重要过程的理解。

Mathews, F.S.MCB-0091084Electron transfer between biological macromolecules is fundamental to many biological processes. It usually occurs within a transient complex in which the participating redox centers and the intervening protein are optimally configured to promote and regulate electron flow between the centers. Such complexes are difficult to study structurally since they are intrinsically unstable and usually cannot be crystallized. Methylamine dehydrogenase (MADH), amicyanin and cytochrome c551i from Paracoccus denitrificans form one of the best characterized physiological electron transfer complexes and is the only complex of three soluble redox proteins for which a high resolution structure is available. MADH is an .2 .2 heterodimer of 124 kDa and contains the unusual redox cofactor tryptophan tryptophylquinone (TTQ). Amicyanin is a blue copper protein of 12.5 kDa which interacts specifically with MADH. Cytochrome c551i is a 17.5 kDa protein which can accept electrons from amicyanin and transfer them via another cytochrome to a terminal oxidase. Aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis is similar to MADH in size and quaternary structure and also contains TTQ as it redox cofactor. However, its specific electron acceptor is an azurin and neither MADH nor AADH will efficiently reduce the alternative acceptor protein, azurin nor amicyanin, respectively. The aims of this proposal are (1) to refine the structures of oxidized and reduced amicyanin at both pH 5.5 and 8.0 at atomic resolution (1 .or below), (2) carry out structural studies of additional redox and pH variants and catalytic intermediates of the MADH/amicyanin and MADH/amicyanin/c551I complexes, (3) structurally characterize mutants of amicyanin and of MADH within these complexes, (4) further analyze redox and cation-bound states of MADH from Methylobacillus flagellatum KT and Methylobacterium extorquens AM1, and (5) complete the structure analysis of AADH and of the binary complex between AADH and azurin.The proposed studies of the MADH and AADH enzyme systems will help identify structural features that are important for electron transfer, including those involved in partner recognition, control of electron transfer and determining paths for electron flow within the complexes. In addition, the structural studies of the mechanism of substrate oxidation by the TTQ cofactor will help shed light on how this cofactor functions in vivo and how it differs from other, more common redox cofactors. TTQ is unusual because it is obtained directly from the fusion of two amino acid side chains coded by genomic DNA rather than from a separate biosynthetic pathway. The MADH and AADH systems are well suited to provide an understanding of these important processes at the molecular level.