SPECTROSCOPIC AND MECHANISTIC STUDIES OF HEME ENZYMES

血红素酶的光谱和机理研究

• 批准号: 6196204
• 负责人: JOHN H DAWSON
• 金额: $ 20.01万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6196204
• 关键词: bioimaging /biomedical imaging; chemical models; circular magnetic dichroism; cytochrome P450; cytochrome c peroxidase; enzyme mechanism; heme oxygenase; hemoprotein; iron sulfur protein; ligands; myoglobin; nitric oxide synthase; peroxidases

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.

描述：（改编自申请人的摘要）本提案寻求支持 用于血红素铁酶的光谱和机理研究项目。 将追求三个重要目标。首先，蛋白质的一个基本方面 结构将被测试：Fe-S键保留在所有氧化态， 细胞色素P450和氯过氧化物酶，但只有在铁的状态， 巯基连接的肌红蛋白（Mb）和细胞色素c过氧化物酶（CCP）突变体。的 导致巯基连接丧失的因素可能与活性组中的相同。 血红素蛋白质的位点，其天然失去巯基连接， 还原在分子模拟的指导下，Mb和CCP双重/三重 将用氢键供体氨基酸制备硫醇盐连接的突变体 被定位以稳定硫醇盐配体朝向还原和氧代铁基 阵硫醇盐连接的氧代铁基加合物的形成将提供简单的 为这些重要国家树立榜样。第二，利用紫外可见/近红外磁性 圆二色性（MCD）光谱将扩展到轴向配体 血红素和二氢卟酚铁蛋白鉴定。血红素酶无处不在 生物分子;每一个的功能都受到其轴向的显著影响。 配体。在一个新的血红素蛋白轴向配体鉴定一直是一个 学习的第一线。MCD光谱学已经发现了很大的应用， 这一目的，但有潜力大大扩大其效用。 为此，将制备大量轴向配体加合物， 涉及先前未被MCD仔细检查的配体组合。接下来，MCD将 用于解决β-半胱氨酸的关键协调结构问题 合成酶、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机理 通过一氧化氮合酶（NOS）和P450激活。氧化亚铁（未特指），稳定 在低温下，将首次用作起点， 解决具体的机械问题。对于P450，假定的过氧铁 将表征用D251 N突变体报告的中间体。最后， 巯基连接血红素氧亚铁态光谱实验 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。