Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

生物大分子之间的电子转移通常发生在一个瞬态复合体中，其中参与的氧化还原中心和干预的蛋白质被优化配置以促进和调节中心之间的电子流动。只有少数这样的配合物结晶了。甲胺脱氢酶（MADH）和氨基青霉素（amicyanin）一起或与细胞色素形成两种具有结构特征的生理电子传递复合物；芳香胺脱氢酶（AADH）与蓝蛋白的配合物包括第三种。MADH和AADH以及醌血红蛋白胺脱氢酶（QHNDH）催化伯胺的氧化脱胺。这三种酶都含有氧化还原辅助因子色氨酸醌（TRQ），色氨酸侧链含有两个醌氧原子。在MADH和AADH中，TRQ与另一种色氨酸交联或与QHNDH中的半胱氨酸交联。本项目的目的是开展以下结构研究：(1)单独或与MADH复合氧化还原特性改变的氨基花青素突变体；(2)AADH/azurin复合物的氧化还原变异体和配体结合形式；(3)TRQ及其与半胱氨酸的交联在QHNDH中的作用和生物发生。结果将有助于确定生物电子转移调制的重要结构特征，包括伴侣识别，驱动力调节，重组能量和门控。由于翻译后修饰的氨基酸侧链在生物学中无处不在，其作用和生物发生机制尚不清楚，因此对TRQ生物发生的研究具有重要意义。拟议的活动所产生的更广泛的影响将包括促进国内和国际研究方面的本科培训。将继续努力使当地本科生参与结构研究，意大利的电子转移络合物的单晶光谱研究将为国外学生提供培训环境。这项研究还将通过与日本和意大利的科学家及其学生建立国际伙伴关系，加强研究基础设施。

Electron transfer between biological macromolecules 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. Only a few such complexes have been crystallized. Methylamine dehydrogenase (MADH) and amicyanin, both together and with a cytochrome, form two structurally characterized physiological electron transfer complexes; the complex of aromatic amine dehydrogenase (AADH) with azurin comprises a third. MADH and AADH, as well as quinohemoprotein amine dehydrogenase (QHNDH), catalyze the oxidative deamination of primary amines. All three enzymes contain the redox cofactor tryptophylquinone (TRQ), a tryptophan side chain containing two quinone oxygen atoms. TRQ is crosslinked to another tryptophan in MADH and AADH or to a cysteine in QHNDH. The aims of this project are to carry out structural studies of (1) amicyanin mutants with altered redox properties, alone and in complex with MADH, (2) redox variants and ligand-bound forms of the AADH/azurin complex and (3) the roles and biogenesis of TRQ and its crosslink to cysteine in QHNDH. The results will help identify structural features important for modulation of biological electron transfer, including partner recognition, regulation of driving force, reorganization energy and gating. The studies of TRQ biogenesis are important because of the ubiquity in biology of posttranslationally modified amino acid side chains whose roles and biogenic mechanisms are poorly understood. The broader impacts resulting from the proposed activity will include the promotion of undergraduate training in research both nationally and internationally. Efforts to involve local undergraduates in the structural studies will continue and single crystal spectroscopic studies of electron transfer complexes in Italy will provide a training environment for students abroad. The research will also enhance research infrastructure through international partnerships with scientists and their students in Japan and Italy.