Spectroelectrochemistry of Flavo and Metalloproteins

黄素和金属蛋白的光谱电化学

• 批准号: 6642089
• 负责人: MARIAN T. STANKOVICH
• 金额: $ 25.45万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6642089
• 关键词: Raman spectrometry; X ray crystallography; acyl coA dehydrogenases; allosteric site; electrochemistry; enzyme activity; enzyme mechanism; enzyme substrate; enzyme substrate complex; ferredoxin; flavoproteins; metalloproteins; methane monooxygenase; miscellaneous oxidoreductase; nuclear magnetic resonance spectroscopy; oxidation reduction reaction; oxidoreductase; protein structure function; ribonucleotide reductase; thermodynamics

Electron transfer between protein partners (e.g., substrates, products, other proteins, and small effector molecules) is a key component of many important biological processes. Kinetic and thermodynamic studies suggest that the electron transfer of many enzymes (and thus their overall reaction mechanisms) is regulated by the binding of protein partners. The acyl-CoA dehydrogenases (ACDs) that play a major role in b-oxidation, and ribonucleotide reductase (RNR), which provides deoxynucleotides for DNA synthesis, are both involved in electron transfers that are highly regulated by protein partner binding. The redox properties of their noncovalently-bound protein cofactors (flavins and diiron centers, respectively) have been found to be excellent indicators of the thermodynamic changes linked to regulation, since substrate/product binding perturbs the midpoint potentials of these reporter groups. Thus, spectroelectrochemistry can often identify changes that cannot be observed by any other means. The objective of this proposal is to gain additional insight into the interactions involved in protein partner binding that result in the catalytic regulation of the ACDs and RNR. For the ACDs, our goal is to learn how the interactions induced by ligand binding affect catalysis and the thermodynamic properties of both the ACD and the ligand. A wealth of thermodynamic data show that the ACDs are highly regulated. Recent Raman spectroscopic and redox studies using the same Raman active product analog have led us to hypothesize that substrate binding causes the redox potential of the enzyme (E) to change, and that in turn, binding to the enzyme causes the substrate (S) to be polarized in the active site, demonstrating that the properties of the activated complex [E.S] are different from those of the free E and S. In order to learn more about the activated complex [E.S], we have devised a new series of substrate analogs to probe that species, further exploring substrate polarization and changes in redox chemistry. For RNR, we propose to probe the interactions responsible for the gating of the long-range electron transfer between subunits. Redox potential studies of the iron center have provided the only data indicating that R1 binding affects R2. The R2 iron center acts as a reporter group for conformational changes and/or long- range interactions that result from the formation of the [R1.R2] complex, and we expect to observe additional changes caused by the binding of substrate, product, and allosteric effectors.

蛋白质伴侣（例如底物、产物、其他蛋白质和小效应分子）之间的电子转移是许多重要生物过程的关键组成部分。 动力学和热力学研究表明，许多酶的电子转移（以及它们的整体反应机制）受到蛋白质伴侣的结合的调节。 在 b-氧化中起主要作用的酰基辅酶 A 脱氢酶 (ACD) 和为 DNA 合成提供脱氧核苷酸的核糖核苷酸还原酶 (RNR) 都参与受蛋白质伴侣结合高度调节的电子转移。 已发现其非共价结合的蛋白质辅因子（分别为黄素和二铁中心）的氧化还原特性是与调节相关的热力学变化的极好指标，因为底物/产物结合扰乱了这些报告基团的中点电位。 因此，光谱电化学通常可以识别任何其他方法无法观察到的变化。 该提案的目的是进一步了解蛋白质伴侣结合中涉及的相互作用，从而导致 ACD 和 RNR 的催化调节。对于 ACD，我们的目标是了解配体结合诱导的相互作用如何影响 ACD 和配体的催化和热力学性质。 大量的热力学数据表明 ACD 受到高度监管。最近使用相同拉曼活性产物类似物的拉曼光谱和氧化还原研究使我们假设底物结合导致酶 (E) 的氧化还原电位发生变化，反过来，与酶的结合导致底物 (S) 在活性位点极化，证明活化复合物 [E.S] 的性质与游离 E 和 S 的性质不同。 复合物[E.S]，我们设计了一系列新的底物类似物来探测该物种，进一步探索底物极化和氧化还原化学的变化。对于 RNR，我们建议探究负责亚基之间长程电子转移门控的相互作用。铁中心的氧化还原电位研究提供了唯一表明 R1 结合影响 R2 的数据。 R2 铁中心充当构象变化和/或长程相互作用的报告基团，这些变化是由 [R1.R2] 复合物的形成引起的，我们期望观察到由底物、产物和变构效应子的结合引起的额外变化。