title: Role of the Proximal Cysteine Hydrogen Bonding Interaction in Cytochrome P450 2B4 Studied by Cryoreduction, Electron Paramagnetic Resonance, and Electron-Nuclear Double Resonance Spectroscopy.
abstract: 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 (14)N but has relatively little effect on the (1)H 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 S = (3)/2 state with the anion radical exchange-coupled to the Fe(II). In the mutant, it localizes on the iron, generating an S = (1)/2 Fe(I) state. (iii) The additional H-bond has little effect on g values and (1)H-(14)N 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.
authors.affiliation: Department of Chemistry, Northwestern University , 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.affiliation: Department of Anesthesiology, University of Michigan, and VA Medical Center , 2215 Fuller Road, Ann Arbor, Michigan 48105, United States.
authors.forename: Sangchoul
authors.initials: S
authors.lastname: Im
authors.affiliation: Department of Chemistry, Northwestern University , 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors.forename: Muralidharan
authors.initials: M
authors.lastname: Shanmugam
authors.affiliation: Department of Chemistry, Northwestern University , 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors.forename: William A
authors.initials: WA
authors.lastname: Gunderson
authors.affiliation: Department of Anesthesiology, University of Michigan, and VA Medical Center , 2215 Fuller Road, Ann Arbor, Michigan 48105, United States.
authors.forename: Naw May
authors.initials: NM
authors.lastname: Pearl
authors.affiliation: Department of Chemistry, Northwestern University , 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.affiliation: Department of Anesthesiology, University of Michigan, and VA Medical Center , 2215 Fuller Road, Ann Arbor, Michigan 48105, United States.
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000378:metabolism*; D015925:Cryopreservation; D018360:Crystallography, X-Ray / Q000379:methods*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006860:Hydrogen Bonding
chemical_list: D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D003545:Cysteine