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Content archived on 2024-04-30

The kinetics and reaction mechanisms of metal proteins that can react with oxygen


There are several proteins containing metal-sites that can react with oxygen known yet, but the detailed mechanisms of these reactions are mostly still in the dark. These reactions include normally intermediates or products with unpaired electrons, and these can be detected by electron paramagnetic resonance (EPR) spectroscopy and investigated in detail by electron nuclear double resonance (ENDOR) spectroscopy. ENDOR/EPR will be used to study the reactions of ribonucleotide reductases (RNR produces deoxyribonucleotides -> DNA, pharmacy), tyrosine and phenylalanine hydroxylases (TyrH and PheH, -> phenylketonuria, pharmacy), alkene monooxygenase (AMO, -> stereospecific epoxides, chemistry) and particulate methane monooxygenase (pMMO, oxidises small (halogen)carbohydrates, bioremediation). RNR contains a stable tyrosyl radical (Y-) close to a diiron centre essential for enzymatic activity which detailed structure in RNR from different sources will be addressed. Y. is produced in a reaction with oxygen, the kinetics and intermediates of which will be further investigated. In case of the hydroxylases the question of the amounts and structure of the ligating histidines will be addressed by ENDOR studies of apoenzyme and enzyme-substrate complexes. About the metal-sites in AMO and pMMO very little is known, so that all kinds of studies suitable will be performed on them. If necessary, other suitable techniques, like high field EPR (245 GHz, collaboration with A.-L. Barra, Grenoble, France), will be used. Time resolved X-ray crystallography on RNR is planned in collaboration with P. Nordlund (Stockholm) using caged substances to follow the structural changes of intermediates (synchrotron beam time at the Swiss-Norwegian beam-line at ESRF Grenoble is available). Training content (objective, benefit and expected impact) I am experienced in electron paramagnetic resonance (EPR) and the metal protein ribonucleotide reductase. I studied the kinetics of various enzymes by means of divers kinetic techniques, including cryoenzymology. Training in electron nuclear double resonance (ENDOR) will enable me to reveal the detailed structure of paramagnetic species (intermediates and products) in proteins which can react with oxygen. ENDOR facilities are not available in my present place and Prof. Andersson i well experienced in this technique. Further I will extend my knowledge on metalloproteins by studies on tyrosine and phenylalanine hydroxylases as well as alkene monooxygenase and particulate methane monooxygenase. This will lead to detailed reaction mechanisms which will be compared between the different proteins. Thus, I will learn new techniques that I am currently not using and gain insight into new metalloproteins and their structure-function relationships. Links with industry / industrial relevance (22)

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University of Oslo
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0316 Oslo

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