The Use of Deuterated Camphor as a Substrate in ¹H ENDOR Studies of Hydroxylation by Cryoreduced Oxy P450cam Provides New Evidence of the Involvement of Compound I

Electron paramagnetic resonance and ¹H electron nuclear double resonance (ENDOR) spectroscopies have been used to analyze intermediate states formed during the hydroxylation of (1<i>R</i>)-camphor (H₂-camphor) and (1<i>R</i>)-5,5-dideuterocamphor (D₂-camphor) as induced by cryoreduction (77 K) and annealing of the ternary ferrous cytochrome P450cam–O₂–substrate complex. Hydroxylation of H₂-camphor produced a primary product state in which 5-<i>exo</i>-hydroxycamphor is coordinated with Fe­(III). ENDOR spectra contained signals derived from two protons [Fe­(III)-bound C5-OH_<i>exo</i> and C5-H_<i>endo</i>] from camphor. When D₂-camphor was hydroxylated under the same condition in H₂O or D₂O buffer, both ENDOR H_<i>exo</i> and H_<i>endo</i> signals are absent. For D₂-camphor in H₂O buffer, H/D exchange causes the C5-OH_<i>exo</i> signal to reappear during relaxation upon annealing to 230 K; for H₂-camphor in D₂O, the magnitude of the C5-OH_<i>exo</i> signal decreases via H/D exchange. These observations clearly show that Compound I is the reactive species in the hydroxylation of camphor in P450cam

Spectroscopic and Crystallographic Evidence for the Role of a Water-Containing H‑Bond Network in Oxidase Activity of an Engineered Myoglobin

Heme-copper oxidases (HCOs) catalyze efficient reduction of oxygen to water in biological respiration. Despite progress in studying native enzymes and their models, the roles of non-covalent interactions in promoting this activity are still not well understood. Here we report EPR spectroscopic studies of cryo­reduced oxy-F33Y-Cu_BMb, a functional model of HCOs engineered in myoglobin (Mb). We find that cryo­reduction at 77 K of the O₂-bound form, trapped in the conformation of the parent oxy­ferrous form, displays a ferric-hydro­peroxo EPR signal, in contrast to the cryo­reduced oxy-wild-type (WT) Mb, which is unable to deliver a proton and shows a signal from the peroxo-ferric state. Crystallography of oxy-F33Y-Cu_BMb reveals an extensive H-bond network involving H₂O molecules, which is absent from oxy-WTMb. This H-bonding proton-delivery network is the key structural feature that transforms the reversible oxygen-binding protein, WTMb, into F33Y-Cu_BMb, an oxygen-activating enzyme that reduces O₂ to H₂O. These results provide direct evidence of the importance of H-bond networks involving H₂O in conferring enzymatic activity to a designed protein. Incorporating such extended H-bond networks in designing other metallo­enzymes may allow us to confer and fine-tune their enzymatic activities

Compound I Is the Reactive Intermediate in the First Monooxygenation Step during Conversion of Cholesterol to Pregnenolone by Cytochrome P450scc: EPR/ENDOR/Cryoreduction/Annealing Studies

Cytochrome P450scc (CYP11A1) catalyzes conversion of cholesterol (CH) to pregnenolone, the precursor to all steroid hormones. This process proceeds via three sequential monooxygenation reactions: two stereospecific hydroxylations with formation first of 22<i>R</i>-hydroxycholesterol (22-HC) and then 20α,22<i>R</i>-dihydroxycholesterol (20,22-DHC), followed by C20–C22 bond cleavage. Herein we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the first hydroxylation step by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc-cholesterol complex. This approach is fully validated by the demonstration that the cryoreduced ternary complex of oxy-P450scc-CH is catalytically competent and hydroxylates cholesterol to form 22-HC with no detectable formation of 20-HC, just as occurs under physiological conditions. Cryoreduction of the ternary complex trapped at 77 K produces predominantly the hydroperoxy-ferriheme P450scc intermediate, along with a minor fraction of peroxo-ferriheme intermediate that converts into a new hydroperoxo-ferriheme species at 145 K. This behavior reveals that the distal pocket of the parent oxy-P450scc-cholesterol complex exhibits an efficient proton delivery network, with an ordered water molecule H-bonded to the distal oxygen of the dioxygen ligand. During annealing of the hydroperoxy-ferric P450scc intermediates at 185 K, they convert to the primary product complex in which CH has been converted to 22-HC. In this process, the hydroperoxy-ferric intermediate decays with a large solvent kinetic isotope effect, as expected when proton delivery to the terminal O leads to formation of Compound I (Cpd I). ¹H ENDOR measurements of the primary product formed in deuterated solvent show that the heme Fe­(III) is coordinated to the 22<i>R</i>-O¹H of 22-HC, where the ¹H is derived from substrate and exchanges to D after annealing at higher temperatures. These observations establish that Cpd I is the agent that hydroxylates CH, rather than the hydroperoxy-ferric heme

Electron Paramagnetic Resonance and Electron-Nuclear Double Resonance Studies of the Reactions of Cryogenerated Hydroperoxoferric–Hemoprotein Intermediates

The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein–O₂ complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H₂O₂ and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferric intermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferric intermediates decay with measurable rates at temperatures above 170 K with appreciable solvent kinetic isotope effects. The hydroperoxoferric intermediate of β-chains converts to the <i>S</i> = 3/2 Cpd I, which in turn decays to an electron paramagnetic resonance (EPR)-silent product at temperature above 220 K. For all the other hemoproteins studied, cryoannealing of the hydroperoxo intermediate directly yields an EPR-silent majority product. In each case, a second follow-up 77 K γ-irradiation of the annealed samples yields low-spin EPR signals characteristic of cryoreduced ferrylheme (compound II, Cpd II). This indicates that in general the hydroperoxoferric intermediates relax to Cpd I during cryoanealing at low temperatures, but when this state is not captured by reaction with a bound substrate, it is reduced to Cpd II by redox-active products of radiolysis

Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22­(<i>R</i>)-hydroxycholesterol (HC) and then 20α,22­(<i>R</i>)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20–C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) <i>J. Am. Chem. Soc. 134</i>, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170–180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and ¹H ENDOR measurements of the primary product formed in D₂O demonstrate that Cpd I is the active species. They show that the primary product contains Fe­(III) coordinated to the 20-O¹H of DHC with the ¹H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone

Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron–Nuclear Double Resonance Spectroscopy

Crystallographic studies have shown that the F429H mutation of cytochrome P450 2B4 introduces an H-bond between His429 and the proximal thiolate ligand, Cys436, without altering the protein fold but sharply decreases the enzymatic activity and stabilizes the oxyferrous P450 2B4 complex. To characterize the influence of this hydrogen bond on the states of the catalytic cycle, we have used radiolytic cryoreduction combined with electron paramagnetic resonance (EPR) and (electron–nuclear double resonance (ENDOR) spectroscopy to study and compare their characteristics for wild-type (WT) P450 2B4 and the F429H mutant. (i) The addition of an H-bond to the axial Cys436 thiolate significantly changes the EPR signals of both low-spin and high-spin heme-iron­(III) and the hyperfine couplings of the heme-pyrrole ¹⁴N but has relatively little effect on the ¹H ENDOR spectra of the water ligand in the six-coordinate low-spin ferriheme state. These changes indicate that the H-bond introduced between His and the proximal cysteine decreases the extent of S → Fe electron donation and weakens the Fe­(III)–S bond. (ii) The added H-bond changes the primary product of cryoreduction of the Fe­(II) enzyme, which is trapped in the conformation of the parent Fe­(II) state. In the wild-type enzyme, the added electron localizes on the porphyrin, generating an <i>S</i> = ³/₂ state with the anion radical exchange-coupled to the Fe­(II). In the mutant, it localizes on the iron, generating an <i>S</i> = ¹/₂ Fe­(I) state. (iii) The additional H-bond has little effect on <i>g</i> values and ¹H–¹⁴N hyperfine couplings of the cryogenerated, ferric hydroperoxo intermediate but noticeably slows its decay during cryoannealing. (iv) In both the WT and the mutant enzyme, this decay shows a significant solvent kinetic isotope effect, indicating that the decay reflects a proton-assisted conversion to Compound I (Cpd I). (v) We confirm that Cpd I formed during the annealing of the cryogenerated hydroperoxy intermediate and that it is the active hydroxylating species in both WT P450 2B4 and the F429H mutant. (vi) Our data also indicate that the added H-bond of the mutation diminishes the reactivity of Cpd I

Enzymatic and Cryoreduction EPR Studies of the Hydroxylation of Methylated <i>N</i>^ω‑Hydroxy‑l‑arginine Analogues by Nitric Oxide Synthase from <i>Geobacillus stearothermophilus</i>

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and NO in a two-step process involving the intermediate <i>N</i>^ω-hydroxy-l-arginine (NHA). It was shown that Cpd I is the oxygenating species for l-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N^ω-OH and N^ω-H of NHA significantly inhibits the conversion of NHA into NO and l-citrulline by mammalian NOS. Kinetic studies now show that N^ω-methylation of NHA has a qualitatively similar effect on H₂O₂-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N^ω-hydroxy l-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA’s, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of l-citrulline with the intensity of the <b>g 2.26</b> EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of l-citrulline in these experiments and <i>k</i>_obs for the H₂O₂-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N^ω-OH and N^ω-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase