X-RAY ABSORPTION SPECTROSCOPY OF FERRYL INTERMEDIATES

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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).