title: Kinetic isotope effects on the rate-limiting step of heme oxygenase catalysis indicate concerted proton transfer/heme hydroxylation.
abstract: Heme oxygenase (HO) catalyzes the O2 and NADPH/cytochrome P450 reductase-dependent conversion of heme to biliverdin, free iron ion, and CO through a process in which the heme participates as both dioxygen-activating prosthetic group and substrate. We earlier confirmed that the first step of HO catalysis is a monooxygenation in which the addition of one electron and two protons to the HO oxy-ferroheme produces ferric-alpha-meso-hydroxyheme (h). Cryoreduction/EPR and ENDOR measurements further showed that hydroperoxo-ferri-HO converts directly to h in a single kinetic step without formation of a Compound I. We here report details of that rate-limiting step. One-electron 77 K cryoreduction of human oxy-HO and annealing at 200 K generates a structurally relaxed hydroperoxo-ferri-HO species, denoted R. We here report the cryoreduction/annealing experiments that directly measure solvent and secondary kinetic isotope effects (KIEs) of the rate-limiting R --> h conversion, using enzyme prepared with meso-deuterated heme and in H2O/D2O buffers to measure the solvent KIE (solv-KIE), and the secondary KIE (sec-KIE) associated with the conversion. This approach is unique in that KIEs measured by monitoring the rate-limiting step are not susceptible to masking by KIEs of other processes, and these results represent the first direct measurement of the KIEs of product formation by a kinetically competent reaction intermediate in any dioxygen-activating heme enzyme.The observation of both solv-KIE(298) = 1.8 and sec-KIE(298) = 0.8 (inverse) indicates that the rate-limiting step for formation of h by HO is a concerted process: proton transfer to the hydroperoxo-ferri-heme through the distal-pocket H-bond network, likely from a carboxyl group acting as a general acid catalyst, occurring in synchrony with bond formation between the terminal hydroperoxo-oxygen atom and the alpha-meso carbon to form a tetrahedral hydroxylated-heme intermediate. Subsequent rearrangement and loss of H2O then generates h.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Davydov
authors.forename: Toshitaka
authors.initials: T
authors.lastname: Matsui
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Fujii
authors.forename: Masao
authors.initials: M
authors.lastname: Ikeda-Saito
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
mesh_terms: D002384:Catalysis; D041961:Deuterium Exchange Measurement; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D006900:Hydroxylation; D007700:Kinetics; D011522:Protons
chemical_list: D011522:Protons; D006418:Heme; D006419:Heme Oxygenase (Decyclizing)