Iron-sulfur cofactors involved in metal center assembly and catalysis of hydrogenase

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In the second funding period of the Priority Program (PP) 1927, our joint project will focus, first, on the elucidation of the unusual active site properties of an O2-tolerant, thermostable NAD+-reducing [NiFe]-hydrogenase and their impact on the catalytic properties of this complex FeS cluster enzyme. The molecular and mechanistic details will be investigated in a collaborative approach, which will involve electron paramagnetic resonance (EPR), infrared (IR), and resonance Raman (RR) spectroscopy to identify and characterize catalytically relevant redox states both in vitro and in vivo. Comparative studies will be performed on a related F420-reducing [NiFe]-hydrogenase. In this context, we will apply a recently established experimental setup, which allows (cryogenic) IR and RR measurements on the same protein crystal under controlled gas atmospheres. In combination with X-ray crystallography and theoretical methods, these experiments will provide detailed structural and electronic information on individual catalytic intermediates. This interdisciplinary approach will be extended to analyze [Fe] hydrogenase and nitrogenase. In the second part of our joint project, we seek to improve the understanding of the multistep biosynthesis process of the NiFe(CN-)2(CO) cofactor of [NiFe] hydrogenase. We will investigate the reaction mechanism by which HypX converts formyl-tetrahydrofolate via formyl-CoA into the active site CO ligand of [NiFe] hydrogenases under oxic conditions. Experiments are planned to unveil the composition of the FeS cluster-containing HypCD maturation complex competent in receiving the (HypX-generated) CO molecule to eventually form the Fe(CN-)2(CO) unit. Based on a recently developed strategy, we are now able to purify isolated large subunits of [NiFe]-hydrogenases in different maturation stages of the catalytic center. This unique situation allows us to unravel the sequence of events of NiFe(CN-)2(CO) cofactor assembly. Vibrational spectroscopic techniques will be used to investigate the role and interplay of FeS centers and other metals in the maturation and catalysis of hydrogenase, nitrogenase(-like) enzymes and Complex I. In close collaboration with members of the PP consortium and external partners, we will exploit our results to gain a detailed picture of the overarching principles of FeS-based maturation and catalysis.