SPECTROELECTROCHEMISTRY OF FLAVO AND METALLOPROTEINS

许多基本的生物过程都涉及电子转移 在蛋白质伙伴之间或在蛋白质伙伴之间 多组分酶复合体的电子转移 催化中心可能与化学反应偶联。举例 这两种类型的电子转移都包括光合作用的减少。 NADP到NADPH；脱氧核糖核苷酸的合成； 甲烷的氧化；和(3)脂肪酸的氧化。在每一个中 上述情况，最近的热力学和在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，因此整个反应机制，是 受蛋白质配对、酶组分、 或基材/产品。我们发现，它的氧化还原性质 蛋白质的辅因子是很好的变化报告组 与调节亚基结合时在蛋白质中发生的 底物；有时用这种方法观察到变化，它 不能用光谱观察到。从我们的氧化还原研究来看，我们有 发现了两个非常不同和有趣的监管案例 与化学反应相耦合的电子转移反应。首先，一个 束缚过渡态模拟，它与产品非常相似 中链乙酰辅酶A氧化脂肪酰辅酶A酯的研究 脱氢酶(MCAD)，被发现通过以下方式调节反应 适当地改变其氧化还原电势。相比之下，衬底 甲烷单加氧酶(MMO)的活性不起作用 酶的催化成分；更确切地说是与还原酶的结合 在监管部门的出席下，产生了深刻的 对MMO的热力学和动力学产生了前所未有的影响。 我们在下一个资助期的目标是在我们的发现基础上 L)提供拉曼光谱证据 活性中心中底物/产物的假设极化 MCAD.这些光谱研究将提供证据证明 提出的过渡态，并使我们能够阐明其机制 在分子水平上。2)检验假设，即电子 R_1和R_2组分之间的转移反应 核糖核苷酸还原酶(RNR)可能受这两个复合体的调控 RI和R2之间的形成及RI中的底物结合 变构结合介导的进一步调控的亚基 效应器。3)通过使用酰辅酶A去饱和酶，建立了新的全球 为酶的调节机制提供证据 在其他系统中也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.