Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations

Carsten Krebs, Laura M.K. Dassama, Megan L. Matthews, Wei Jiang, John C. Price, Victoria Korboukh, Ning Li, Joseph M. Bollinger, Jr.

Research output: Contribution to journalReview article

14 Citations (Scopus)

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 (<2mM) of the co-substrate, O 2, 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 [O 2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3mM 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 O 2 and (2) the in situ generation of O 2 from 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 O 2 across 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. The in situ approach permits generation of a pulse of at least 5mM O 2 within ∼1ms and accumulation of O 2 to effective concentrations of up to ∼11mM (i.e. ∼10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.

Original languageEnglish (US)
Pages (from-to)234-243
Number of pages10
JournalCoordination Chemistry Reviews
Volume257
Issue number1
DOIs
StatePublished - Jan 1 2013

Fingerprint

chlorite dismutase
Enzymes
Oxygenases
Oxygenation
Molecular oxygen
Substrates
Dilution
enzymes
Oxidoreductases
Solubility
Gases
Oxidation
Kinetics
oxygenation
oxidase
Liquids
half life
dilution
purity
solubility

All Science Journal Classification (ASJC) codes

  • Physical and Theoretical Chemistry
  • Inorganic Chemistry
  • Materials Chemistry

Cite this

Krebs, Carsten ; Dassama, Laura M.K. ; Matthews, Megan L. ; Jiang, Wei ; Price, John C. ; Korboukh, Victoria ; Li, Ning ; Bollinger, Jr., Joseph M. / Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations. In: Coordination Chemistry Reviews. 2013 ; Vol. 257, No. 1. pp. 234-243.
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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 (<2mM) of the co-substrate, O 2, 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 [O 2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3mM 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 O 2 and (2) the in situ generation of O 2 from 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 O 2 across 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. The in situ approach permits generation of a pulse of at least 5mM O 2 within ∼1ms and accumulation of O 2 to effective concentrations of up to ∼11mM (i.e. ∼10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.",
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Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations. / Krebs, Carsten; Dassama, Laura M.K.; Matthews, Megan L.; Jiang, Wei; Price, John C.; Korboukh, Victoria; Li, Ning; Bollinger, Jr., Joseph M.

In: Coordination Chemistry Reviews, Vol. 257, No. 1, 01.01.2013, p. 234-243.

Research output: Contribution to journalReview article

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T1 - Novel approaches for the accumulation of oxygenated intermediates to multi-millimolar concentrations

AU - Krebs, Carsten

AU - Dassama, Laura M.K.

AU - Matthews, Megan L.

AU - Jiang, Wei

AU - Price, John C.

AU - Korboukh, Victoria

AU - Li, Ning

AU - Bollinger, Jr., Joseph M.

PY - 2013/1/1

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N2 - 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 (<2mM) of the co-substrate, O 2, 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 [O 2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3mM 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 O 2 and (2) the in situ generation of O 2 from 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 O 2 across 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. The in situ approach permits generation of a pulse of at least 5mM O 2 within ∼1ms and accumulation of O 2 to effective concentrations of up to ∼11mM (i.e. ∼10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.

AB - 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 (<2mM) of the co-substrate, O 2, 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 [O 2] (and therefore on the concentration of reactive intermediate(s) that can be rapidly accumulated) of ∼1-1.3mM 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 O 2 and (2) the in situ generation of O 2 from 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 O 2 across 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. The in situ approach permits generation of a pulse of at least 5mM O 2 within ∼1ms and accumulation of O 2 to effective concentrations of up to ∼11mM (i.e. ∼10-fold greater than by the conventional approach). The use of these new techniques for studies of oxygenases and oxidases is discussed.

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