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时复合体中，参与其中的氧化还原中心和中间蛋白质被优化配置以促进和调节中心之间的电子流动。只有几个这样的络合物被结晶。甲胺脱氢酶(MADH)和氨蓝蛋白与细胞色素一起形成两个结构特征的生理电子转移复合体；芳香胺脱氢酶(AADH)与天青素形成第三个复合体。MADH和AADH以及藜红蛋白胺脱氢酶(QHNDH)催化伯胺的氧化脱氨。这三种酶都含有氧化还原辅因子色氨酸(TRQ)，这是一个含有两个苯醌氧原子的色氨酸侧链。TRQ在MADH和AADH中与另一种色氨酸或在QHNDH中与半胱氨酸交联。本项目的目的是开展以下方面的结构研究：(1)氧化还原特性改变的氨基蓝蛋白突变体，单独或与MADH形成复合体，(2)AADH/天青复合体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体在QHNDH中的作用和生物发生。这些结果将有助于识别对生物电子传递的调节重要的结构特征，包括伙伴识别、驱动力的调节、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普遍存在，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。拟议活动产生的更广泛影响将包括促进国内和国际的本科生研究培训。将继续努力让当地本科生参与结构研究，意大利电子转移络合物的单晶光谱研究将为海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来加强研究基础设施。

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.