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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 O2. These enzymes are important in nature for the recovery of large amounts of carbon from aromatic compounds. The catalytic site contains either Fe or Mn coordinated by a facial triad of two His and one Glu or Asp residues. Previous studies have shown that Fe(II) and Mn(II) can be interchanged in enzymes from different organisms to catalyze similar substrate reactions. In combination, quantitative electron paramagnetic resonance spectroscopy and rapid freeze-quench experiments allow us to follow the concentrations of four different Mn species, including key metal intermediates in the catalytic cycle, as the enzyme turns over its natural substrate. Two intermediates are observed: a Mn(III)-radical species which is either Mn-superoxide or Mn-substrate radical, and a unique Mn(II) species which is involved in the rate-limiting step of the cycle and may be Mn-alkylperoxo.

SUBMITTER: Gunderson WA

PROVIDER: S-EPMC2806810 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Electron paramagnetic resonance detection of intermediates in the enzymatic cycle of an extradiol dioxygenase.

Gunderson William A WA Zatsman Anna I AI Emerson Joseph P JP Farquhar Erik R ER Que Lawrence L Lipscomb John D JD Hendrich Michael P MP

Journal of the American Chemical Society 20081008 44

Extradiol catecholic dioxygenases catalyze the cleavage of the aromatic ring of the substrate with incorporation of both oxygen atoms from O2. These enzymes are important in nature for the recovery of large amounts of carbon from aromatic compounds. The catalytic site contains either Fe or Mn coordinated by a facial triad of two His and one Glu or Asp residues. Previous studies have shown that Fe(II) and Mn(II) can be interchanged in enzymes from different organisms to catalyze similar substrate ...[more]

PMID: 18839948

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Project description: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 S = 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.

Project description:The extradiol dioxygenases are a large subclass of mononuclear nonheme Fe enzymes that catalyze the oxidative cleavage of catechols distal to their OH groups. These enzymes are important in bioremediation, and there has been significant interest in understanding how they activate O2. The extradiol dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) provides an opportunity to study this process, as two O2 intermediates have been trapped and crystallographically defined using the slow substrate 4-nitrocatechol (4NC): a side-on Fe-O2-4NC species and a Fe-O2-4NC peroxy bridged species. Also with 4NC, two solution intermediates have been trapped in the H200N variant, where H200 provides a second-sphere hydrogen bond in the wild-type enzyme. While the electronic structure of these solution intermediates has been defined previously as FeIII-superoxo-catecholate and FeIII-peroxy-semiquinone, their geometric structures are unknown. Nuclear resonance vibrational spectroscopy (NRVS) is an important tool for structural definition of nonheme Fe-O2 intermediates, as all normal modes with Fe displacement have intensity in the NRVS spectrum. In this study, NRVS is used to define the geometric structure of the H200N-4NC solution intermediates in HPCD as an end-on FeIII-superoxo-catecholate and an end-on FeIII-hydroperoxo-semiquinone. Parallel calculations are performed to define the electronic structures and protonation states of the crystallographically defined wild-type HPCD-4NC intermediates, where the side-on intermediate is found to be a FeIII-hydroperoxo-semiquinone. The assignment of this crystallographic intermediate is validated by correlation to the NRVS data through computational removal of H200. While the side-on hydroperoxo semiquinone intermediate is computationally found to be nonreactive in peroxide bridge formation, it is isoenergetic with a superoxo catecholate species that is competent in performing this reaction. This study provides insight into the relative reactivities of FeIII-superoxo and FeIII-hydroperoxo intermediates in nonheme Fe enzymes and into the role H200 plays in facilitating extradiol catalysis.

Dataset Information

Electron paramagnetic resonance detection of intermediates in the enzymatic cycle of an extradiol dioxygenase.

Publications

Electron paramagnetic resonance detection of intermediates in the enzymatic cycle of an extradiol dioxygenase.

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