Protein preparations in frozen solution and in single crystals were investigated by various techniques of Electron Spin Resonance (ESR) spectroscopy and electron-nuclear double resonance (ENDOR) experiments. One could thus obtain a detailed insight into the electronic structure and ligand binding situation of the enzyme’s catalytic site in several intermediate steps. Furthermore, using modern relativistic Density Functional Theory (DFT) calculations, it was possible to accurately calculate the spectroscopic observables and compare them with those measured experimentally. For the first time it was feasible to treat systems as complicated and big as that of an enzymatic active centre.

Thus, a direct structure-to-function relationship for the active centre during the catalytic process could be established and revealed the atomic composition of the active centre in every step, the substrate binding site and the reaction mechanism. The fine-tuning of the reaction mechanism by the protein environment and the protein cofactors was also investigated and showed nature’s sophistication in constructing natural catalysts.

The project was performed at the Max Volmer Laboratory for Biophysical Chemistry at the Technical University Berlin under the supervision of Prof. Wolfgang Lubitz. The international and interdisciplinary project involved research groups from Europe and Japan and was a co-operation between protein structural chemists, microbiologists, physicists, spectroscopists and theoretical chemists. The project was supported by the Fonds der Chemischen Industrie and the Deutsche Forschungsgemeinschaft (DFG). Matthias Stein is now working with the anterio consult & research GmbH, Mannheim, on computer-aided drug design and all aspects of scientific computing.