SPECTROSCOPIC AND MECHANISTIC STUDIES OF HEME ENZYMES

描述：(改编自申请人的摘要)本提案寻求支持 有关血红素铁酶的光谱和机理研究的计划。 我们将追求三个重要目标。首先，蛋白质的一个基本方面 将测试结构：Fe-S键在所有的氧化态中都保持不变 细胞色素P450和氯过氧化物酶，但仅在铁的状态下 硫酸盐连接肌红蛋白(Mb)和细胞色素c过氧化物酶(CCP)突变体。这个 导致硫酸盐连接丧失的因素可能与活性相同。 天然失去硫酸盐连接的血红素蛋白的位置 还原。以分子模拟为指导，Mb和Ccp双/三 用氢键供体氨基酸制备硫代连接突变体 定位为使硫酸盐配体稳定在还原和氧化铁基上 队形。硫酸酯连接的氧铁加合物的形成将提供简单的 这些重要州的模型。第二，使用紫外可见/近红外磁性 圆二色谱(MCD)将扩展到轴向配体 血红素和氯化铁蛋白的鉴定。血红素酶无处不在 生物分子；每个分子的功能都受到其轴向的显著影响 配基。一种新的血红素蛋白的轴向配基鉴定一直是 学习的第一条线。MCD光谱学已被广泛应用于 这一目的，但有可能显著扩大其用途。 为此，将制备大量的轴向配体加合物 涉及MCD以前没有仔细研究过的配基组合。接下来，MCD将 用于解决胱硫酮β的关键配位结构问题 合酶、血红素加氧酶、可溶性鸟苷酸环化酶和含铁氯 系统。第三个目标是研究分子氧的作用机理。 由一氧化氮合酶(NOS)和P450激活。氧化亚铁，稳定的一氧化氮合酶 在低温下，将首次被用作起点 解决具体的机械问题。在P450中，推定的过氧铁 将对报道的带有D251N突变体的中间体进行表征。最后， 硫酸盐连接亚铁血红素含氧态的光谱实验 蛋白质及其单电子还原产物将显著增加 我们对这些重要但知之甚少的血红素状态的了解。

DESCRIPTION: (adapted from applicant's abstract) This proposal seeks support for a program of spectroscopic and mechanistic studies of heme iron enzymes. Three important goals will be pursued. First, a fundamental aspect of protein structure will be tested: the Fe-S bond is retained in all oxidation states of cytochrome P450 and chloroperoxidase but only in ferric states of thiolate-ligated myoglobin (Mb) and cytochrome c peroxidase (CCP) mutants. The factors that lead to loss of thiolate ligation may be the same as in the active sites of the heme proteins that naturally lose thiolate ligation upon reduction. Guided by molecular modeling, Mb and CCP double/triple thiolate-ligated mutants will be prepared with H-bond donor amino acids positioned to stabilize the thiolate ligand toward reduction and oxoferryl formation. Formation of thiolate-ligated oxoferryl adducts would provide simple models for these important states. Second, use of UV-visible/near-IR magnetic circular dichroism (MCD) spectroscopy will be extended for axial ligand identification in heme and chlorin iron proteins. Heme enzymes are ubiquitous biomolecules; the function of each is significantly influenced by its axial ligands. Axial ligand identification in a new heme protein is always one of the first lines of study. MCD spectroscopy has already found great application for this purpose, but there is the potential to significantly extend its utility. Toward this end, a large number of axial ligand adducts will be prepared that involve ligand combinations not previously scrutinized by MCD. Next, MCD will be used to address key coordination structure issues for cystathione beta synthase, heme oxygenase, soluble guanylyl cyclase and iron chlorin-containing systems. The third goal is to study the mechanism of molecular oxygen activation by nitric oxide synthase (NOS) and P450. Oxyferrous NOS, stabilized at low temperatures, will be used for the first time as the starting point to address specific mechanistic questions. With P450, the putative peroxyferric intermediate reported with the D251N mutant will be characterized. Finally, spectroscopic experiments on oxyferrous states of thiolate-ligated heme proteins and their one-electron reduced products will significantly increase our knowledge of these important, but poorly understood heme states.