SPECTROELECTROCHEMISTRY OF FLAVO AND METALLOPROTEINS

许多重要的生物过程都涉及电子转移 蛋白质伙伴之间或内部蛋白质伙伴之间 多组分酶复合物，其中电子转移 催化中心可以与化学反应耦合。的例子 两种类型的电子转移都包括光合作用还原 NADP 至 NADPH；脱氧核糖核苷酸的合成；的 甲烷的氧化；和脂肪酸的（3-氧化）。在每个 上述情况，最近的热力学和某些情况下的动力学 对重要蛋白质成分的测量表明 电子转移以及整个反应机制是 受蛋白质伴侣、酶成分的结合调节， 或基材/产品。我们发现氧化还原性质 蛋白质的辅因子是极好的变化报告基团 结合调节亚基后发生在蛋白质中或 基材；有时用这种方法观察到变化 无法通过光谱观察到。根据我们的氧化还原研究，我们有 发现了两个非常不同且有趣的监管案例 电子转移反应与化学反应耦合。首先，一个 结合过渡态类似物，与产品非常相似 中链酰基辅酶A对脂肪酰基辅酶A酯的氧化作用 脱氢酶（MCAD）被发现可以通过以下方式调节反应： 适当改变其氧化还原电位。相比之下，基材 甲烷单加氧酶 (MMO) 在激活 酶的催化成分；而是还原酶的结合 在监管部门的存在下，产生了深远的影响 对MMO的热力学和动力学产生了前所未有的影响。 我们下一个资助期的目标是在我们的发现的基础上再接再厉 通过以下方式： l) 提供拉曼光谱证据 活性位点中底物/产物的假设极化 的MCAD。这些光谱研究将为 提出的过渡状态并使我们能够阐明其机制 在分子水平上。 2) 检验电子的假设 RI和R2组分之间的转移反应 核糖核苷酸还原酶（RNR）可能由两种复合物门控 RI 和 R2 之间的形成以及 RI 中的底物结合 具有由变构结合介导的进一步控制的亚基 效应器。 3) 通过使用酰基辅酶A去饱和酶，建立一个新的全球 通过提供证据来研究酶的调节机制 在其他系统中观察到调节蛋白的调节 除了MMO和RNR。

Many essential biological processes involve electron-transfer either between protein partners or between protein partners within multicomponent enzyme complexes where electron transfer at the catalytic center may be coupled to chemical reactions. Examples of both types of electron transfer include the photosynthetic reduction of NADP+ to NADPH; the synthesis of deoxyribonucleotides; the oxidation of methane; and the (3-oxidation of fatty acids. In each of the above cases, recent thermodynamic and in some cases kinetic measurements of the important protein components have suggested that electron transfer, and thus the overall reaction mechanisms, are regulated by the binding of protein partners, enzyme components, or substrates/products. We have found that the redox properties of the cofactors of proteins are excellent reporter groups for changes occurring in a protein upon binding of regulatory subunits or substrates; sometimes changes are observed with this method which cannot be observed spectrally. From our redox studies, we have discovered two very different and interesting cases of regulation of electron transfer reactions coupled to chemical reactions. First, a bound transition state analog, which closely resembled the product of fatty acyl-CoA ester oxidation by medium chain acyl-CoA dehydrogenase (MCAD), was found to regulate the reaction by appropriately shifting its redox potential. In contrast, the substrate of methane monooxygenase (MMO) plays no role in activating the catalytic component of the enzyme; rather the binding of reductase in the presence of the regulatory component had a profound and unprecedented effect on the thermodynamics and kinetics of MMO. Our goal in the next grant period is to build on our discoveries in the following ways: l) Provide Raman spectroscopic evidence for the hypothesized polarization of substrate/product in the active site of MCAD. These spectroscopic studies will provide evidence for a proposed transition state and enable us to elucidate the mechanism on the molecular level. 2) Test the hypothesis that the electron transfer reaction between the RI and R2 components of ribonucleotide reductase (RNR) is likely gated by both complex formation between RI and R2 and substrate binding in the RI subunit with further control mediated by the binding of allosteric effectors. 3) By using Acyl-CoA Desaturase, establish a new global regulation mechanism for enzymes by providing evidence that regulation by regulatory proteins is observed in other systems besides MMO and RNR.