O 2-evolving chlorite dismutase as a tool for studying O 2-utilizing enzymes

Laura M.K. Dassama, Timothy H. Yosca, Denise A. Conner, Michael H. Lee, Béatrice Blanc, Bennett R. Streit, Michael T. Green, Jennifer L. DuBois, Carsten Krebs, J. Martin Bollinger

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30 Scopus citations


The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas's modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O 2 at concentrations far exceeding 2 mM. This method was used to define the O 2 concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme's Mn IV/Fe III cofactor forms from a Mn II/Fe II complex and O 2 via a Mn IV/Fe IV intermediate, at effective O 2 concentrations as high as ∼10 mM. With a more soluble receptor, myoglobin, an O 2 adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C-H-bond-cleaving Fe IV-oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo-Fe 2 III/III complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe 2 III/IV intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O 2 evolution permits a >5 mM "pulse" of O 2 to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O 2 addition steps.

Original languageEnglish (US)
Pages (from-to)1607-1616
Number of pages10
Issue number8
StatePublished - Feb 28 2012

All Science Journal Classification (ASJC) codes

  • Biochemistry


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