44624-AC3
Understanding C-H Bond Activation in Oxidative Heme Proteins
Background X-ray absorption measurements on the thiolate-ligated iron(IV)oxo (ferryl) in chloroperoxidase compound II (CPO-II) have suggested a role for thiolate ligation in Nature's hydroxylating heme enzymes. These measurements indicated an Fe-O bond of 1.82 Å for this intermediate (pH 6.5). This value is longer than expected for an authentic ferryl unit (typically near 1.65 Å) but in excellent agreement with density functional calculations on a protonated ferryl heme (Fe(IV)-OH, 1.81 Å). The existence of a basic ferryl species has important implications for C-H bond activation. In the consensus hydroxylation mechanism (called the rebound mechanism), a ferryl-radical species called compound I abstracts hydrogen from substrate to form a protonated ferryl (similar to CPO-II), 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 the following equation. D(O-H) = + 23.06 x E0cmpd-I + 1.37 x pKacmpd-II + 57 ± 2 (kcal/mol) This equation 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 P450 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. This project examines the issue of ferryl protonation in heme enzymes. Density functional methods have been used to parameterize Badger's rule (an empirical law relating bond distance and stretching frequency). These calculations are being coupled to experiments (Mössbauer, resonance Raman, and EXAFS) to provide a complete characterization of non-thiolate ligated ferryl species. We specifically seek to determine an upper limit for the ferryl pKa's. Current Status Tim Yosca, a graduate student, is currently working on an important aspect of the project. In order to use the equation outlined above, the ferryl species must be in equilibrium with respect to protonation. Tim is attempting to confirm that this is the case by examining the exchange of the ferryl oxygen with solvent. It is known that the ferryl oxygen of horseradish peroxidase compound II exchanges above pH 5, but the process has not been examined at lower pH. The oxygen exchange has been shown through resonance Raman experiments where HRP-II is prepared with 18O and changes in the ferryl stretching frequency are monitored as the 18O ferryl oxygen is replaced with 16O from the solvent. Tim is performing similar experiments to determine the lowest pH at which he can observe the oxygen exchange. This will allow him to put a lower limit on the ferryl pKa in HRP-II, and determine the D(O-H) for this system. This work will provide considerable insight into C-H bond activation in heme proteins.
