title: Novel Approaches for the Accumulation of Oxygenated Intermediates to Multi-Millimolar Concentrations.
abstract: Metalloenzymes that utilize molecular oxygen as a co-substrate catalyze a wide variety of chemically difficult oxidation reactions. Significant insight into the reaction mechanisms of these enzymes can be obtained by the application of a combination of rapid kinetic and spectroscopic methods to the direct structural characterization of intermediate states. A key limitation of this approach is the low aqueous solubility (< 2 mM) of the co-substrate, O2, which undergoes further dilution (typically by one-third or one-half) upon initiation of reactions by rapid-mixing. This situation imposes a practical upper limit on [O2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3 mM in such experiments as they are routinely carried out. However, many spectroscopic methods benefit from or require significantly greater concentrations of the species to be studied. To overcome this problem, we have recently developed two new approaches for the preparation of samples of oxygenated intermediates: (1) direct oxygenation of reduced metalloenzymes using gaseous O2and (2) thein situgeneration of O2from chlorite catalyzed by the enzyme chlorite dismutase (Cld). Whereas the former method is applicable only to intermediates with half lives of several minutes, owing to the sluggishness of transport of O2across the gas-liquid interface, the latter approach has been successfully applied to trap several intermediates at high concentration and purity by the freeze-quench method. Thein situapproach permits generation of a pulse of at least 5 mM O2within ∼ 1 ms and accumulation of O2to effective concentrations of up to ∼ 11 mM (i.e. ∼ 10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 ; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Carsten
authors.initials: C
authors.lastname: Krebs
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Laura M K
authors.initials: LM
authors.lastname: Dassama
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Megan L
authors.initials: ML
authors.lastname: Matthews
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: John C
authors.initials: JC
authors.lastname: Price
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Victoria
authors.initials: V
authors.lastname: Korboukh
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
authors.affiliation: Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 ; Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: J Martin
authors.initials: JM
authors.lastname: Bollinger
keywords: ferryl; intermediate; iron; non-heme; oxygen; peroxo; superoxo