X-RAY ABSORPTION SPECTROSCOPY OF FERRYL INTERMEDIATES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。该研究试图使用x射线吸收光谱，结合穆斯堡尔光谱，来确定（作为pH值的函数）氧化血红素蛋白中发现的一些高价铁（IV）氧或铁酰中间体的质子化状态。这项工作的重要性在于，观察到金属氧氧化物对氢的提取能力与h原子提取过程中形成的O-H键的强度有关。在氧化血红素酶中，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。已知只有硫代酸连接的血红素酶能将氧插入碳氢键。这一反应被认为是通过一种涉及氢提取的机制发生的。最近，我们（MTG）对铁基形式的氯过氧化物酶（一种已知羟化活化C-H键的硫酸连接血红素酶）进行了EXAFS测量，发现该中间体是碱性的（pKa ~ 8.2）。这一发现表明，自然可能是使用硫代酸连接来促进氢的提取，而不是通过组氨酸连接的血红素酶进行单电子氧化。本提案中概述的实验旨在检验这一假设。他们试图1)验证CPO-II中硫代酸连接铁基的基本性质，使用低温还原技术；2)通过检查P450BM-3和P450nor的铁基形式，确定硫代酸连接铁基是否一般是碱性的；3)通过检查几种组氨酸连接铁基的质子化状态作为pH的函数，确定碱性铁基是否是硫代酸连接血红素的独特特征。4)首次提供了硫代酸连接化合物I（在硫代酸连接血红素酶中负责提取氢的高价中间体）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).