Resonance Raman Spectroscopic Studies of Hydroperoxo-Myoglobin at Cryogenic Temperatures

Ibrahim, Mohammed; Denisov, Ilia G.; Makris, Thomas M.; Kincaid, James R.; Sligar, Stephen G.

doi:10.1021/ja036949d

Cited by 59 publications

(77 citation statements)

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“…This shift is somewhat smaller, however, than the ∼45 cm -1 shift observed for hydroperoxo myoglobin, relative to oxy myoglobin. 14 This observation of a trapped hydroperoxo form is quite consistent with behavior expected for the native cytochrome P450cam enzyme, which has previously been shown to proceed directly at 77 K to the hydroperoxo form, with rather small fractions of the unprotonated species (Fe−OO -) being present in the preparations. 7 Similarly, recent crystallographic studies of cryotrapped CPO Compound III also were interpreted to lead to radiation-induced conversion to the hydroperoxo form, with no evidence for a trapped peroxo-ligated species.…”

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“…[11][12][13][14][15] In fact, the feasibility of coupling this powerful spectroscopic probe with cryogenic radiolysis of heme protein samples was recently demonstrated and suggested to be a promising new approach to structurally characterize these important intermediates. 3,14,15 The present work uses RR to provide the first direct observation of the structure-sensitive internal vibrational modes of the (Fe−OOH) fragment of the hydroperoxo−ferric intermediate of the CYP101 enzyme.Samples of the oxygenated form of CYP101 were prepared by bubbling dioxygen gas through 30% glycerol/buffer solutions of the (ferrous) enzyme contained in 5 mm NMR tubes. The tube was shaken for several seconds to ensure efficient mixing and quickly frozen in NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript.…”

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confidence: 99%

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

et al. 2007

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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 ...

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

et al. 2007

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“…Ϫ16°C or less (46). However, I. Morishima and colleagues could not observe the horseradish peroxidase compound 0 intermediate at ambient temperature with a microsecond-resolved absorption spectrometer (47), although resonance Raman evidence for an Fe-OOH fragment in peroxo-bound myoglobin has in fact been reported (34). Difficulty in detecting the compound 0 species might be due to the fact that the substrate hydrogen peroxide already carries the two protons required for catalysis and that the subsequent heterolytic O-O bond cleavage is rapidly catalyzed by the enzyme, forming compound I.…”

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confidence: 98%

Crystal Structures of Chloroperoxidase with Its Bound Substrates and Complexed with Formate, Acetate, and Nitrate

Kühnel

Blankenfeldt

Terner

et al. 2006

Journal of Biological Chemistry

104

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Chloroperoxidase (CPO) is a heme-thiolate enzyme that catalyzes hydrogen peroxide-dependent halogenation reactions. Structural data on substrate binding have not been available so far. CPO was therefore crystallized in the presence of iodide or bromide. One halide binding site was identified at the surface near a narrow channel that connects the surface with the heme. Two other halide binding sites were identified within and at the other end of this channel. Together, these sites suggest a pathway for access of halide anions to the active site. The structure of CPO complexed with its natural substrate cyclopentanedione was determined at a resolution of 1.8 Å . This is the first example of a CPO structure with a bound organic substrate. In addition, structures of CPO bound with nitrate, acetate, and formate and of a ternary complex with dimethylsulfoxide (Me 2 SO) and cyanide were determined. These structures have implications for the mechanism of compound I formation. Before binding to the heme, the incoming hydrogen peroxide first interacts with Glu-183. The deprotonated Glu-183 abstracts a proton from hydrogen peroxide. The hydroperoxo-anion then binds at the heme, yielding compound 0. Glu-183 protonates the distal oxygen of compound 0, water is released, and compound I is formed.

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“…In the first classic HRP step the resting ferric (Fe III ) high spin (S ϭ 5/2) form is oxidized by hydrogen peroxide to a water molecule and a heme state that is two oxidation equivalents higher (compound I) than the resting ferric form. This first step propagates through a hydroperoxointermediate (compound 0) where compound I is generated through a heterolytic cleavage of the O-O bond (11)(12)(13). The distal His is assumed to function as an acid/base catalyst to facilitate heterolytic cleavage by accepting a proton from the inner oxygen (oxygen ligated to the iron) and then donating it to the outer (leaving) oxygen.…”

Section: Myoglobin (Mb)mentioning

confidence: 99%

Crystallographic and Spectroscopic Studies of Peroxide-derived Myoglobin Compound II and Occurrence of Protonated FeIV–O

Hersleth

Uchida

Røhr

et al. 2007

Journal of Biological Chemistry

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Resonance Raman Spectroscopic Studies of Hydroperoxo-Myoglobin at Cryogenic Temperatures

Cited by 59 publications

References 45 publications

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

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

Crystal Structures of Chloroperoxidase with Its Bound Substrates and Complexed with Formate, Acetate, and Nitrate

Crystallographic and Spectroscopic Studies of Peroxide-derived Myoglobin Compound II and Occurrence of Protonated FeIV–O

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