SFB 498 : Bacterial [NiFe]-Hydrogenases: Key Reactions of the Biological Activation of Molecular Hydrogen

Biological cleavage of H2 is a common metabolic process in prokaryotes and lower eukaryotes and is catalyzed by two major classes of enzymes, the [NiFe]- and the [Fe]-hydrogenases. Three distinct [NiFe]-hydrogenases of Ralstonia eutropha (formerly Alcaligenes eutrophus) are in the centre of this project, the regulatory (RH), the NAD-linked (SH) and the membrane-bound (MBH) hydrogenase. The research program is focused on the following aspects:<br>
-Molecular analysis of the initial reactions of the H2-sensing mechanism aims at the identification of a newly identified redox cofactor and its interaction with other functional domains in the RH. Selected RH mutant proteins affected in gas transport, electron and proton transfer are investigated in detail using a wide range of biochemical and spectroscopic techniques. Efforts to isolate RH crystals for X-ray defraction are being continued.<br>
-The experiments on the two energy-converting hydrogenases (SH and MBH) are focused on the characterization of the [NiFe] active site which is modified in the SH by insertion of two extra CN-ligands. This modification will be correlated with the specific properties of the respective hydrogenases in particular with a postulated mechanism for oxygen protection.<br>
-The assembly of the unique [NiFe] cofactor requires a series of maturation steps, mediated by the so-called Hyp proteins. To explore the cofactor and its protein environment in more depth we integrated a new subproject into our working program. The experiments are mainly focused on two questions: (i) Is the HypX protein responsible for the insertion of the extra diatomic ligands and, if yes, is a tetrahydrofolate derivative the C1-donating agent? (ii) Is a single- instead of a multidomain HypF protein sufficient for the insertion of the diatomic ligands? In vivo and in vitro experiments are conducted with the two forms, the HypF1 and the HypF2 proteins of R. eutropha.