Novel bio-synthetic constructs for the analysis and manipulation of the hydrogen catalysis of [FeFe]-hydrogenases

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[FeFe]-hydrogenases are highly efficient H2-producing biocatalysts and therefore promising candidates for blueprints for the development of new synthetic catalysts for H2 production. Unfortunately, essential functional aspects of the active center (H-cluster) and its protein environment are still poorly understood. This applies in particular to the aspects of proton-coupled electron transport in the catalytic mechanisms and the problem of the oxygen sensitivity of this otherwise highly efficient enzyme class. These gaps in comprehension are to be closed here through the complementary use of molecular biology, synthesis chemistry, spectroscopy and theory within the framework of a well-established and proven multidisciplinary cooperation. On the one hand, the mechanistic and spatial differentiation between regulatory and catalytic proton transfer will be pursued on the basis of 25 site directed mutagenesis variants. On the other hand, we will obtain sample series of wild type and mutant variants for the spectroscopic monitoring of the process of O2-dependent H-cluster degradation in a time-resolved "freeze-quench" procedure. The use of 18O2 for H-cluster exposition allows us to track the resulting oxidation products (e.g. CN18O, C18O2 or H218O) via mass-spectrometry. In addition, we will try to increase the O2 resistance of one of the examined enzymes (CpI) by selectively increasing of the molecular sieve effect in its gas channel system, whereby larger variant pools are generated and sampled on this effect. In order to create a comprehensive overall picture, protein-biochemistry, crystallographic, enzyme kinetic and spectroscopic techniques (ATR-FTIR, XAS/XES, NRVS, FTIR, EPR and Mössbauer) as well as quantum mechanical computations are applied in all sub-projects. In addition to the investigations on protein derivatives, element substitutions in the H-cluster architecture will be employed to elucidate the individual functional contributions of all ligands for the reaction cycle and their influence on the reactivity (H+, e-, H2, O2) concerning both sub-clusters, [4Fe]H and [2Fe]H. An exchange of nitrogen in the bridgehead of the dithiolate-ligand against phosphorus could e.g. maintain the proton conductivity to the H-cluster, while avoiding the formation of reactive oxygen species (ROS) under O2 contact. The influence of metal specificity and electron density distribution of the H-cluster for the process of O2-induced H-cluster degradation is also to be investigated, employing novel metal homologs of the [Fe2(ADT)(CO)4(CN)2]2- cofactor to be fitted into the binding niche of [FeFe]-hydrogenases CPI and HydA1 by the in vitro maturation procedure which is a well-established and powerful technique in the ongoing collaboration of both applicants.