An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ).
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.
Wiertz
Frank G M
FG
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.
Richter
Oliver-Matthias H
OM
Cherepanov
Alexey V
AV
MacMillan
Fraser
F
Ludwig
Bernd
B
de Vries
Simon
S
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*
D001426:Bacterial Proteins; D014364:Tryptophan; D045304:Cytochromes c; D010088:Oxidoreductases; C020094:duroquinol oxidase; D010100:Oxygen