| Cytochromes P450 (P450) are thiolate-ligated heme enzymes that play critical roles in many biological processes, including metabolism of xenobiotics. Thiolate-ligated heme enzymes are unique among heme proteins because they catalyze oxygen insertion reactions. Recently, an X-ray absorption spectroscopy study of the ferryl form of chloroperoxidase (CPO-II) suggested a novel role for thiolate-ligation. It revealed that CPO-II was not an authentic ferryl (iron(IV)oxo), but rather a protonated ferryl with a long Fe-O bond length of 1.82 Å (typically ferryls have Fe-O bond lengths near 1.65 Å). This finding has important implications for P450 hydroxylation chemistry where a basic ferryl may provide the driving force to perform hydrogen atom abstraction. The hydroxylation mechanism for P450’s describes two high-valent iron intermediates. These intermediates are highly reactive. Due to almost identical first coordination spheres of chloroperoxidase (CPO) and P450’s, CPO has been utilized as a model to investigate high-valent iron intermediates of P450 reaction chemistry.;To gain insight into the reactive intermediate of P450 enzymatic catalysis, X-ray absorption measurements of chloroperoxidase compound I were performed. CPO-I is an authentic ferryl species (a doubly bonded Fe-O bond with a length of 1.65 Å) and strong axial-ligand interactions result in a long Fe-S bond length of 2.48 Å. This is the first unambiguous structural characterization of a thiolate-ligated compound I species and provides a first glimpse into the reactive intermediate of P450 reaction chemistry.;We have also examined the protonation state of CPO-II with the use of multiple spectroscopies: Mössbauer spectroscopy coupled with density functional calculations, X-ray absorption spectroscopy, ENDOR spectroscopy and resonance Raman spectroscopy. A Badger’s rule analysis of the EXAFS-determined Fe-O bond length of CPO-II (1.82 Å) affords an Fe-O stretching frequency of νBadger=563 cm−1. A deuterium and 18O sensitive stretching frequency, νFe-OH=565 cm −1, was located by resonance Raman spectroscopy in remarkable agreement to what is predicted from Badger’s rule.;All evidence, to date, suggests that the basic ferryls are a general feature of thiolate-ligated heme enzymes. This provides a reasonable explanation of why Nature utilizes the electron donating thiolate ligand to perform some very demanding oxidizing reactions. |
