Comparative Studies of [FeFe]-Hydrogenases

• Applicant: Dr. Sven Stripp
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 552022686

[FeFe]-hydrogenases are water-soluble iron-sulfur enzymes catalyzing hydrogen turnover (H2 <-> 2 H+ + 2 e-) in bacteria and plants. [FeFe]-hydrogenases oxidize H2 as an electron source for the anabolic metabolism, e.g., co-catalyzing the reductive fixation of CO2 into organic carbohydrates like format. In proton reduction direction, H2 is released from the cell to control the redox balance. Advantageous properties like a turnover number of 20.000 H2 s-1 without significant electrical overpotential (-420 mV vs SHE at pH 7) render [FeFe]-hydrogenase an important bioinorganic model system. Understanding the molecular proceedings of hydrogenase will inspire catalytic compounds that can be used in fuel cells. The catalytic cofactor of [FeFe]-hydrogenase comprises of a bimetallic iron center ([FeFe]) that is covalently attached to a [4Fe-4S] cluster ([4Fe]H). The so-called 'H-cluster' carries two cyanide ligands (CN) and up to four carbon monoxide ligands (CO) that anchor the diiron site within the active site fold by hydrogen-bonding contacts and adjust the redox potential. [FeFe]-hydrogenases are excellent targets for Fourier-transform infrared spectroscopy (FTIR) due to the strong dipole moment of the CN and CO ligands that absorb in a frequency regime not overlaid by other IR signals. At the example of various hydrogenases, I developed an in situ technique that allows recording FTIR difference spectra triggered by gas titrations. For this, the enzyme is probed in attenuated total reflection configuration (ATR). Done carefully, a 'humid' gas atmosphere above the protein film can be maintained that prevents desiccation of the sample. The analysis of the catalytic function of [FeFe]-hydrogenases is an important focus of my work since 2017, alongside the development and application of in situ ATR FTIR spectroscopy on gas-processing metalloenzymes in general. Within the framework of this research proposal, I will investigate final aspects of the catalytic mechanism of [FeFe]-hydrogenase. In particular, this includes the pH-dependence of H2 production and proton-coupled electron transfer to the [4Fe]H cluster. Moreover, I will establish serial X-ray diffraction to investigate the crystal structure of reduced [FeFe]-hydrogenase at room temperature. In another work package, I propose a number of experiments that will facilitate investigating the enzymatic coupling between H2-catalysis and electron bifurcation and/or CO2-catalysis.

• DFG Programme: Research Grants