X-RAY ABSORPTION SPECTROSCOPY OF FERRYL INTERMEDIATES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该研究旨在使用X射线吸收光谱，结合穆斯堡尔光谱，以确定（作为pH值的函数）在氧化血红素蛋白中发现的一些高价铁（IV）氧代或铁基中间体的质子化状态。这项工作的重要性在于观察到金属氧合物夺取氢的能力与在夺取H原子期间形成的O-H键的强度成比例。在氧化血红素酶中，这种O-H键的强度取决于化合物I（一种铁基自由基）的单电子还原电位和称为化合物II的铁基自由基的pKa。 已知只有巯基连接的血红素酶将氧插入C-H键。该反应被认为是通过涉及氢提取的机制发生的。最近，我们之一（MTG）对氯过氧化物酶（一种巯基连接的血红素酶，已知可羟基化活化的C-H键）的铁基形式进行了EXAFS测量，发现该中间体是碱性的（pKa约为8.2）。这一发现表明，自然界可能使用硫醇连接来促进氢的提取，而不是由组氨酸连接的血红素酶进行的单电子氧化。 本提案中概述的实验旨在检验这一假设。他们试图1）使用croygenic还原技术验证CPO-II中硫醇盐连接的亚铁的基本性质，2）通过检查P450 BM-3和P450 nor的亚铁形式来确定硫醇盐连接的亚铁是否通常是碱性的，3）确定碱性亚铁是否是硫醇盐连接的亚铁血红素的独特特征，通过检测几种组氨酸连接的铁酰基物质的质子化状态作为pH的函数，和4）首次提供了硫醇盐连接的化合物I的详细几何描述，（在巯基连接的血红素酶中负责氢提取的高价中间体）。

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).