title: An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ).
abstract: The pre-steady state reaction kinetics of the reduction of molecular oxygen catalyzed by fully reduced cytochrome oxidase from Escherichia coli and Paracoccus denitrificans were studied using the newly developed microsecond freeze-hyperquenching mixing-and-sampling technique. Reaction samples are prepared 60 and 200 micros after direct mixing of dioxygen with enzyme. Analysis of the reaction samples by low temperature UV-Vis spectroscopy indicates that both enzymes are trapped in the PM state. EPR spectroscopy revealed the formation of a mixture of two radicals in both enzymes. Based on its apparent g-value and lineshape, one of these radicals is assigned to a weakly magnetically coupled oxo-ferryl tryptophan cation radical. Implications for the catalytic mechanism of cytochrome oxidases are discussed.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.
authors.forename: Frank G M
authors.initials: FG
authors.lastname: Wiertz
authors.forename: Oliver-Matthias H
authors.initials: OM
authors.lastname: Richter
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Cherepanov
authors.forename: Fraser
authors.initials: F
authors.lastname: MacMillan
authors.forename: Bernd
authors.initials: B
authors.lastname: Ludwig
authors.forename: Simon
authors.initials: S
authors.lastname: de Vries
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D005615:Freezing; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D014364:Tryptophan; D045304:Cytochromes c; D010088:Oxidoreductases; C020094:duroquinol oxidase; D010100:Oxygen