Resonance Raman Detection of the Hydroperoxo Intermediate in the Cytochrome P450 Enzymatic Cycle

Abstract

The resonance Raman spectra of the hydroperoxo complex of camphor-bound CYP101 have been obtained by cryoradiolytic reduction of the oxygenated ferrous form that had been rapidly frozen in water/glycerol frozen solution; EPR spectroscopy was employed to confirm the identity of the trapped intermediate. The ν(O−O) mode, appearing at 799 cm -1 , is observed for the first time in a peroxo-heme adduct. It is assigned unambiguously by employing isotopomeric mixtures of oxygen gas containing 50% 16 O 18 O, confirming the presence of an intact O−O fragment. The ν(Fe−O) mode is observed at 559 cm -1 (H2O). Furthermore, both modes shift down by 3 cm -1 , documenting the formulation as a hydroperoxo complex, in agreement with EPR data.NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Society, Vol 129, No. 20 (2007): pg. 6382-6383. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. Journal of the American Chemical 3The cytochromes P450 heme-thiolate enzymes facilitate quite difficult chemical transformations through a multistep reaction cycle that culminates in the generation of a remarkably potent oxidizing species capable of hydroxylating even inert substrates. [1][2][3] Following substrate binding to the resting state ferric enzyme, two sequential one-electron reductions bracketing the binding of molecular oxygen and a subsequent proton delivery step lead to heterolytic O−O bond cleavage and formation of a highly reactive ferryl heme species comparable to the so-called Compound I intermediate of peroxidases. [4][5][6] Thus, key precursors to this critical cleavage reaction are activated heme-bound peroxo and hydroperoxo fragments; that is, (protoporphyrin)Fe(III)(O2 2-) or Fe(III)(O−OH -). A useful approach to access and study these species is to generate and trap the relatively stable oxy-ferrous P450 complex and then to subject the cryotrapped (77 K) sample to radiolytic reduction using radiation from synchrotron, 60 Co γ-ray, or 32 P sources. 7,8 Enzymatic intermediates produced by cryoradiolytic reduction of the oxygenated complex of cytochrome P450cam (CYP101) have been detected by both electronic absorption and EPR spectroscopic methods. 7,9,10 However, critical mechanistic information has been missing, and it is now important to attempt to provide more detailed structural characterization of the active sites of these species.Resonance Raman (RR) spectroscopy is an especially attractive probe of such species, effectively interrogating both the heme macrocycle structure and various iron−ligand fragments. [11][12][13][14][15] In fact, the feasibility of coupling this powerful spectroscopic probe with cryogenic ...

Evidence for a ferryl intermediate in a heme-based dioxygenase

“…1 A; and (ii) our computational studies indicate a homolytic O-O bond scission following the insertion of the terminal atom of the dioxygen into L-Trp (vide infra). It is noteworthy that the O-O mode of the peroxo derivative of heme proteins with thiolate as a proximal ligand (such as P450 and chloroperoxidase) also appears in the 800 cm Ϫ1 spectral window (46,47); the 799 cm Ϫ1 band is not assigned to a peroxo species because, for histidine-coordinated heme proteins, the Fe-O mode of the peroxo species should be detectable at Ϸ617 cm Ϫ1 (48), while the O-O mode is typically Raman silent [albeit the O-O mode of the peroxo derivatives of cobalt-substituted myoglobin has been identified at Ϸ851 cm Ϫ1 (49)]. It is important to note that the compound-II type of ferryl derivatives in general exhibit electronic transition bands similar to that of superoxo-ferric complexes (see ref.…”

Resonance Raman Spectroscopy Reveals pH-Dependent Active Site Structural Changes of Lactoperoxidase Compound 0 and Its Ferryl Heme O–O Bond Cleavage Products

“…Consistent with those results, in the present work dealing with Compound III derivatives at these widely varying pH values, it is seen that for a low pH value of 5.6, two forms of the dioxygen adduct exist, with a dominant one exhibiting a ν(Fe−O) mode at 558 cm −1 and a lower population of a species exhibiting its ν(Fe−O) mode at 534 cm −1 . As in the case of the corresponding ferrous derivatives, raising the pH of the Compound III form leads to a 38,39,42 the ν(Fe−O) stretching mode of these species is usually observed at ∼25−40 cm −1 higher frequency than the corresponding mode of the dioxygen adduct (Compound III) [ Table 1]. It is noted that, for the peroxo and hydroperoxo derivatives of globins and other histidylligated heme proteins, the ν(O−O) modes are not generally enhanced.…”

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

“…These fleeting intermediates of heme enzyme catalysis could not be isolated in ambient conditions, despite numerous attempts. However, being immersed in liquid nitrogen, they are stable for many months and can be studied using various spectroscopic methods, such as EPR (62,65,67,73-76), UV-Vis absorption spectroscopy (58,59,69,70,76-79), resonance Raman (69,71,72,79,80), Mössbauer (67), and EXAFS (81). …”