An oxo-ferryl tryptophan radical catalytic intermediate in cytochrome c and quinol oxidases trapped by microsecond freeze-hyperquenching (MHQ)

Frank G. M. Wiertz, Oliver-Matthias H. Richter, Alexey V. Cherepanov, Fraser MacMillan, Bernd Ludwig, Simon de Vries

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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 μs 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 P 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.
Original languageEnglish
Pages (from-to)127-130
Number of pages4
JournalFEBS Letters
Volume575
Issue number1-3
DOIs
Publication statusPublished - 24 Sep 2004

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