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44624-AC3
Understanding C-H Bond Activation in Oxidative Heme Proteins

Michael T. Green, Pennsylvania State University

Introduction

This project examines the status of ferryl (FeIV=O) protonation in heme proteins using a combination of theoretical and experimental techniques. The importance of this issue lies in its connection to C-H bond activation. Only heme proteins with axial thiolate ligation are known to hydroxylate hydrocarbons. Recent experiments have revealed that the ferryl forms of these thiolate-ligated enzymes are basic, suggesting a role for thiolate ligation in C-H bond activation. In the consensus hydroxylation mechanism, a ferryl-radical species called compound I abstracts hydrogen from substrate to form a protonated ferryl, which subsequently hydroxylates the substrate radical. The ability of metal-oxos to abstract hydrogen has been shown to scale with the strength of the O-H bond formed during H-atom abstraction, D(O-H). This energy is determined by the reduction potential of compound I and the pKa of compound II, as in Eq. 1.

D(O-H) = + 23.06 x E0cmpd-I + 1.37 x pKacmpd-II + 57 ± 2 (kcal/mol) (1)

Equation 1 highlights the importance of the ferryl pKa and suggests that Nature may be using basic thiolate-ligated ferryls to promote hydrogen abstraction (and subsequent hydroxylation) at biologically viable compound I reduction potentials.

This theory rests upon two important assumptions: 1) that the rebound mechanism is operative in hydrocarbon hydroxylations, and 2) that basic ferryls are a general and unique feature of thiolate-ligated hemes. The first of these assumptions appears well founded, as experimental and theoretical investigations continue to support the rebound mechanism. The merit of the second assumption is not as clear. Recent crystallographic studies suggest that other non-thiolate ligated ferryl species may be protonated as well. Spectroscopic measurements, however, paint a different picture, suggesting that these species are authentic iron(IV)oxos. This project seeks to clarify the issue of ferryl protonation in non-thiolate ligated hemes.

Update

In the first phase of the project a theoretical parameterization of Badger's rule has been used to evaluate the reliability of ferryl bond lengths obtained from X-ray structural techniques. Our investigations have revealed that the long Fe-O bond lengths obtained from the crystal structures of the ferryl forms of myoglobin, horseradish peroxidase, cytochrome c peroxidase, and catalase are the hallmarks of ferric hydroxides (i.e. the crystals have been reduced). In the next phase of the project, our theoretical results will be coupled with Mössbauer, resonance Raman, and EXAFS experiments on the ferryl forms of these enzymes. Our preliminary results on ferryl myoglobin have appeared in the literature. Research is being performed currently by Courtney Krest and Timothy Yosca, two second-year graduate students. These students were supported during the summer using funds from the PRF. Courtney is focusing her attention on catalase and horseradish peroxidase, while Tim is concentrating on cytochrome c peroxidase and myoglobin. Both have spent the last year learning how to express and purify protein as well as how to prepare samples via rapid freeze quench techniques. Both students have received training in the required spectroscopic techniques. We have samples of 57Fe enriched myoglobin in hand, and we are in the process of studying the pH dependence of the ferryl stretching frequency, Mössbauer parameters, and Fe-O bond distance. Courtney is in the process of working out conditions for 57Fe enrichment of catalase, and we hope to have samples of 57Fe enriched ferryl catalase ready for experimental characterization by early spring.

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