Electron Paramagnetic Resonance Detection of Intermediates in the Enzymatic Cycle of an Extradiol Dioxygenase

Abstract

Extradiol catecholic dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O 2 . 1 This reaction is a key step in the ability of Nature to reclaim large quantities of carbon sequestered in aromatic compounds. The active sites of these enzymes contain either Mn or Fe, coordinated by a 2-His-1-carboxylate facial triad, which is a common motif of nonheme Fe(II)-containing enzymes that activate dioxygen. 2 Homoprotocatechuate 2,3-dioxygenase from either Brevi-bacterium fuscum or Arthrobacter globiformis catalyzes the ring opening of homoprotocatechuate (HPCA) and contains Fe(II) (FeHPCD) or Mn(II) (MnMndD), respectively, as the native metals. Structures of FeHPCD from crystals that had been incubated with the substrate analogue 4-nitrocatechol and then exposed to low concentrations of O 2 identified three intermediate complexes in the catalytic cycle: semiquinone substrate radical-Fe superoxo (E-SQ), Fe-alkylperoxo (E-AP), and the Fesemialdehyde ring-opened product (EP). 3 Bond length analysis suggests that the iron remains ferrous in each species. A mechanistic proposal developed from these intermediates and earlier studies suggests that electron transfer from the substrate to O 2 , via the metal to form the E-SQ intermediate, results in simultaneous activation of both substrate and oxygen. Recent work has shown that FeHPCD and MnMndD can each be prepared with the nonphysiological metal in the active site (MnHPCD and FeMndD) and that all four forms have approximately the same catalytic parameters. 4 The fact that FeHPCD and MnHPCD also have superimposable structures suggests that the bound metals retain their inherent ~0.7 V difference in redox potential, leading to the proposal that the metal does not undergo a redox state change in the reaction cycle.Here we explore this question through direct electron paramagnetic resonance (EPR) detection and quantitative analysis of the metal oxidation states of MnHPCD as it turns over the natural substrate, HPCA. MnHPCD was chosen for study because it is available in a pure state, it is EPR active, and its full length form is structurally characterized. Four distinct Mn species are observed, two of which are short-lived intermediates. Our advances in EPR simulation software 5, 6 allow characterization of the electronic environment of all Mn species and the determination of concentrations of all species during turnover. Based on this analysis, a lowconcentration intermediate appearing immediately after O 2 addition is found to contain Mn (III) coupled to a radical. Thus, it is possible that the rapid electron transfer to form the reactive Figure 1. The substrate-free enzyme (E, Figure 1A) shows a six-line hyperfine pattern centered at g = 2.00, with splitting of a = 89 G, and a broad feature at g = 2.52. The hyperfine constant is indicative of a Mn(II) species, and the simulation of this spectrum (spectrum A) is calculated for a single protein-bound Mn(II) species using the parameters given in the Figure 1 cap...

Preparation and characterization of metal-substituted carotenoid cleavage oxygenases

“…Despite their phylogenic diversity, these enzymes share a common catalytic theme: aromatic alkene bond cleavage of a redox-active substrate via direct substrate coordination of the metal center through one or two phenolic oxygen atoms. Both enzymes are thought to use their metal cofactor to channel electrons from the organic substrate to bound dioxygen resulting in simultaneous activation of both substrates with only a transient change in metal redox state [22, 25, 54], which explains the ability of cobalt to support catalysis in these enzymes.…”

Reaction landscape of a pentadentate N5-ligated MnII complex with O2˙− and H2O2 includes conversion of a peroxomanganese(iii) adduct to a bis(μ-oxo)dimanganese(iii,iv) species

“…, superoxide, hydrogen peroxide, and water). 1–3 For example, Mn-dependent dioxygenases 4,5 and oxalate oxidase 6,7 react with molecular oxygen to perform substrate oxidations. Manganese superoxide dismutase 3,8,9 and manganese catalase 2 react with superoxide and hydrogen peroxide, respectively, as defence against reactive oxygen species.…”

Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme

“…tion of an oxidized metal center may occur (23 Ongoing rapid freeze quench (RFQ) studies of the WT 2,3-HPCD with HPCA as a substrate and the native Fe II in the active site have not revealed accumulation of an Fe III -superoxo species that can be detected by EPR or Mössbauer experiments. These results suggest that if such a species is formed, it is very shortlived, so a strategy to slow the reaction is required to detect the putative ferric-superoxo intermediate.…”