Deciphering catalytic mechanism of [FeFe]-hydrogenase by neutron crystallography

• Applicant: Jifu Duan, Ph.D.
• Subject Area: Structural Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 461338801

[FeFe]-hydrogenases catalyze the reversible conversion of protons (H+), electrons and dihydrogen (H2) at their catalytic cofactor, the H-cluster, very efficiently. In recent years, progress has been achieved in elucidating the catalytic mechanism of [FeFe]-hydrogenases with the aid of X-ray crystallography, spectroscopic techniques, electrochemistry and theoretical methods. Many redox states have been identified and characterized, focussing on the characterization of the electron configuration of the H-cluster in the different states. In contrast, the fate of catalytically important hydrogen species has hardly been addressed, which is mostly since hydrogens are very difficult to detect. Within this project I aim to directly analyze the hydrogens of [FeFe]-hydrogenase, especially those that bind to the H-cluster and are involved in proton transfer pathways. Methodically, I will use neutron crystallography and X-ray crystallography combined with selective enrichment of [FeFe]-hydrogenase redox states and their monitoring by in-crystal Fourier-transform infrared (FTIR) spectroscopy.I already have the X-ray crystallography for CpI, [FeFe]-hydrogenase I from Clostridium pasteurianum, established. To extend the use of CpI crystals for neutron diffraction experiments, large CpI single crystals (>0.1 mm3) have to be obtained. Very recently, this very challenging goal has been achieved, paving a solid basis for development of project. The high-quality crystals will be employed to achieve high-resolution X-ray crystal structures in parallel. To enrich and stabilize redox states in the crystals, especially those in which the participation of hydrogens is assumed, enzyme variants will be employed that are trappable in certain states. These will be variants of the polypeptide such as those affected in the proton transfer pathways as well as H-cluster variants, which can be chemically synthesized and incorporated. Most of the desired redox states will be obtained by chemical treatment of the crystals, such as a pH shift or reduction by dithionite. Crystal FTIR will be employed to monitor the establishment of these states at the single crystal level. The large sized-crystals adjusted to individual states will be subjected to neutron diffraction experiments at BIODIFF. The obtained diffraction data will be processed and used for refinements in order to locate the hydrogens within the structures. Unequivocal assignment of hydrogens will allow to characterize the H-cluster configuration in each redox state, and thus may finally provide a full picture of the reaction mechanism of [FeFe]-hydrogenases.

• DFG Programme: Research Grants