High field EPR and ENDOR of [Fe]-Hydrogenase

• Applicant: Dr. Edward Reijerse
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: from 2002 to 2006
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5382067

Hydrogenases are enzymes found in microorganisms which catalyze the simple redox reaction: H2>2H+ + 2e. Two main classes of hydrogenases have been identified: the Iron-Nickel group and the Iron-only group. In the last few years enzymes representative of both groups have been crystallized and their X-ray structures have been determined. The active centre of both classes contains a unique bi-metallic (either Fe-Ni or Fe-Fe) centre with non-protein ligands (CN, CO). The catalytic mechanism of these enzymes is still poorly understood at present. Electron Paramagnetic Resonance (EPR) as well a Electron Nuclear Double Resonance (ENDOR) have proven to be excellent techniques for studying the paramagnetic states of hydrogenases. We intend to study [Fe]-hydrogenase using the most advanced EPR techniques available, i.e. using high magnetic fields and mm-wave frequencies (100-200 GHz). The main advantages will be: a) the very high absolute sensitivity (~108 spins/Gauss.sec) enabling the study of small protein single crystals, and b) the very high Zeeman-resulution leading to single crystal like orientation selection in ENDOR experiments. The main goal of our project is to unravel the sequence of events leading to the heterolytic splitting of hydrogen in the active site. This will be accomplished by identifying the proton-accepting base using the photo labile CO inhibited form of [Fe]-hydrogenase. High field EPR and ENDOR studies will provide a complete picture of the electronic and geometrical structure of the active site in various intermediate states. DFT calculations, calibrated by the experimentally obtained magnetic interactions will further refine our understanding of the catalytic mechanism.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1051: Hochfeld-EPR in Biologie, Chemie und Physik