X-RAY ABSORPTION SPECTROSCOPY OF FERRYL INTERMEDIATES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项研究试图使用X射线吸收光谱，结合穆斯堡尔光谱，来确定(作为pH的函数)一些高价铁(IV)氧代或铁基的质子化状态，这些中间体存在于氧化的血红素蛋白中。这项工作的重要性在于观察到金属氧原子提取氢的能力与在H原子提取过程中形成的O-H键的强度成比例。在氧化型血红素酶中，这种O-H键的强度取决于化合物I(一种铁基自由基物种)的单电子还原电位和被称为化合物II的铁基物种的pKa。只有硫酸盐连接的血红素酶才能将氧插入C-H键。这一反应被认为是通过一个涉及氢提取的机制发生的。最近，我们中的一位(MTG)对一种铁基形式的氯过氧化物酶(一种硫酸盐连接的血红素酶，已知能羟化活化的C-H键)进行了EXAFS测量，发现这种中间体是碱性的(pKa~8.2)。这一发现表明，大自然可能正在使用硫酸盐连接来促进氢的提取，而不是组氨酸连接的血红素酶执行的单电子氧化。这项提案中概述的实验旨在检验这一假设。他们试图1)使用色原还原技术验证CPO-II中硫醇连接铁基的基本性质，2)通过检查P450BM-3和P450NOR的铁基形式来确定硫醇连接铁基是否为一般碱性铁基，3)通过研究几个组氨酸连接铁基物种的质子化状态作为pH的函数来确定碱性铁是否是硫醇连接亚铁的独有特征，以及4)首次提供硫醇连接化合物I(负责硫醇连接血红素酶中氢提取的高价中间体)的详细几何描述。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The research seeks to use x-ray absorption spectroscopy, in combination with Mossbauer spectroscopy, to determine (as a function of pH) the protonation state of a number of high-valent iron(IV)oxo, or ferryl, intermediates found in oxidative heme-proteins. The importance of this work lies in the observation that the ability of metal-oxos to abstract hydrogen scales with the strength of the O-H bond formed during H-atom abstraction. In oxidative heme-enzymes the strength of this O-H bond depends upon the one electron reduction potential of compound I (a ferryl-radical species) and the pKa of the ferryl species, called compound II. Only thiolate-ligated heme enzymes are known to insert oxygen into C-H bonds. This reaction is thought to occur through a mechanism that involves hydrogen abstraction. Recently one of us (MTG) performed EXAFS measurements on a ferryl form of chloroperoxidase (a thiolate-ligated heme-enzyme known to hydroxylate activated C-H bonds) and found that this intermediate was basic (pKa ~ 8.2). This finding suggests that Nature may be using thiolate-ligation to promote hydrogen abstraction rather than the one-electron oxidations performed by histidine-ligated heme-enzymes. The experiments outlined in this proposal aim to examine this hypothesis. They seek to 1) verify the basic nature of the thiolate-ligated ferryl in CPO-II, using croygenic reduction techniques, 2) determine if thiolate-ligated ferryls are basic in general, by examining the ferryl forms of P450BM-3 and P450nor, 3) determine if basic ferryls are a unique feature of thiolate-ligated hemes, by examining the protonation state of several histidine-ligated ferryl species as a function of pH, and 4) provide for the first time a detailed geometrical description of a thiolate-ligated compound I (the high-valent intermediate responsible for hydrogen abstraction in thiolate-ligated heme-enzymes).