Identification of the Fe−O−O Bending Mode in Oxycytochrome P450cam by Resonance Raman Spectroscopy

Macdonald, Iain D. G.; Sligar, Stephen G.; Christian, James F.; Unno, Masashi; Champion, P. M.

doi:10.1021/ja9810383

Cited by 58 publications

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“…For reasons described below, it was decided to include samples prepared from socalled "scrambled" mixtures of O2 isotopomers, such as [1 Figure S1), showing a positive feature at 546 cm -1 for the former and at 515 cm -1 for the latter, frequencies near those previously observed for these adducts in solution at 4 °C. 17,18 It is also noted that these lowered frequencies, relative to corresponding values observed near 570 cm -1 for hemoglobin and myoglobin, are also consistent with expectations based on consideration of the trans-axial ligand effects, as has been carefully considered by Babcock and coworkers. 20 These frozen oxygenated samples were then irradiated with a 60 Co γ-source to produce the reduced hydroperoxo forms; the EPR spectra confirmed the conversion with approximately 60% yield ( Figure S2).…”

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confidence: 86%

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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confidence: 86%

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

et al. 2007

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“…The similarity of these values may be surprising given the large difference in the electron donating ability of the two axial ligands to the heme iron (thiolate for P450cam and imidazole for myoglobin), but the O-O stretching frequencies estimated for oxymyoglobin (∼1131 cm -1 ) (59) and measured for oxyP450cam (1140 cm -1 ) (7) indicate a very similar O-O bond order in the two complexes. Frequencies and bandwidths of the other oxygencentered vibrational modes were also similar in these proteins (7).…”

Section: Solvent Isotopementioning

confidence: 62%

Mechanism of O₂ Activation by Cytochrome P450cam Studied by Isotope Effects and Transient State Kinetics

et al. 2006

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The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.

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“…Similar superoxide character of the heme-bound dioxygen is found in the oxy-derivatives of P450 and CPO. In P450s, as well as their model complexes, the O-O stretching mode is found in the 1139-1147 cm À1 region as summarized in Table 2 [88][89][90][91][92]. Similarly, CPO has a split O-O stretching mode with components at 1137 and 1124 cm À1 (Dr. Ryu Makino of Rikko University, private communication).…”

Section: Heme-oxy I and Heme-oxy Iimentioning

confidence: 99%

Ligand?protein interactions in nitric oxide synthase

Rousseau

Couture

et al. 2005

Journal of Inorganic Biochemistry

117

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Identification of the Fe−O−O Bending Mode in Oxycytochrome P450cam by Resonance Raman Spectroscopy

Cited by 58 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

Mechanism of O₂ Activation by Cytochrome P450cam Studied by Isotope Effects and Transient State Kinetics

Ligand?protein interactions in nitric oxide synthase

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Identification of the Fe−O−O Bending Mode in Oxycytochrome P450cam by Resonance Raman Spectroscopy

Cited by 58 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

Mechanism of O2 Activation by Cytochrome P450cam Studied by Isotope Effects and Transient State Kinetics

Ligand?protein interactions in nitric oxide synthase

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Mechanism of O₂ Activation by Cytochrome P450cam Studied by Isotope Effects and Transient State Kinetics