EPR spectroscopy

Throughout his career, EPR has played an important role as a biophysical technique to gain information about radicals, radical pairs, triplet states and metal centers in chemistry and biochemistry.^[1][11][5] Particular emphasis has been placed on methods that are able to resolve the electron-nuclear hyperfine couplings between the electron spin and the nuclear spins. Next to the more established techniques, electron spin echo modulation (ESEEM) and electron-nuclear double resonance (ENDOR), his group further developed and used electron-electron double resonance- (ELDOR) detected NMR (EDNMR) at a range of mw frequencies.^[12][13][14] These techniques have been used by him and his group to extensively study bacterial photosynthetic reaction centres, their donor-acceptor model complexes, photosystem I, photosystem II,^[1][5] and a number of different hydrogenases.^[11][4]

Oxygen-evolving Complex

During his early career, bacterial photosynthetic reaction centres and oxygenic photosystem I and photosystem II^[1] have been a main focus. He and his group studied light-induced chlorophyll donor^[2] and quinone acceptor radical ions^[3] of the primary electron-transfer chain. Later his research focused on the water splitting cycle (S-states) of photosystem II using advanced multifrequency pulse EPR, ENDOR and EDNMR techniques. His group was able to detect and characterize the flash-generated, freeze-trapped paramagnetic states S₀, S₂ and S₃ (S₁ is diamagnetic and S₄ is a transient state) of the Mn₄Ca₁O_x catalytic cluster. By a careful spectral analysis–backed up by quantum chemical calculations the site oxidation and spin states of all Mn ions and their spin coupling for all intermediates of the catalytic cycle could be detected.^[15][16][17] Further work using advanced Pulse EPR techniques, such as EDNMR, has led to information on the binding of water^[18] and a proposal of an efficient O-O bond formation in the final state of the cycle.^[15][6]

[NiFe]- and [FeFe]-hydrogenase

Extensive work was performed on the [NiFe]-Hydrogenase where the magnetic tensors were measured and related to quantum chemical calculations.^[11][4] Through his work, the structures of all intermediates in the activation path and catalytic cycle of [NiFe]-hydrogenases were obtained. In the course of this work a 0.89 Ångström resolution X-ray crystallography diffraction model of [NiFe]-hydrogenase was achieved.^[19]

Similar work has been accomplished for the [FeFe]-hydrogenases.^[4] A key contribution of his research was the EPR spectroscopic evidence of an azapropane-dithiolate-ligand (ADT-ligand) in the dithiol bridge of the [FeFe]-hydrogenase active site ^[20] and the determination of the magnitude and orientation of the g-tensor using single crystal EPR.^[21] The ADT-ligand was later confirmed by artificial maturation of [FeFe]-hydrogenases.^[22] Using artificial maturation, the protein could be generated without the co-factor (apoprotein) using E. coli mutagenesis and a synthetically created active site could be inserted,^[22][23][24] which has opened new vistas in hydrogenase research.^[25]