Kinetic Isotope Effects on the Rate-Limiting Step of Heme Oxygenase Catalysis Indicate Concerted Proton Transfer/Heme Hydroxylation

Roman Davydov, Toshitaka Matsui, Hiroshi Fujii, Masao Ikeda-Saito, Brian M. Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

72 Scopus citations


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-α-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 α-meso carbon to form a tetrahedral hydroxylated-heme intermediate. Subsequent rearrangement and loss of H2O then generates h.

Original languageEnglish (US)
Pages (from-to)16208-16209
Number of pages2
JournalJournal of the American Chemical Society
Issue number52
StatePublished - Dec 31 2003

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


Dive into the research topics of 'Kinetic Isotope Effects on the Rate-Limiting Step of Heme Oxygenase Catalysis Indicate Concerted Proton Transfer/Heme Hydroxylation'. Together they form a unique fingerprint.

Cite this