An in vitro system for the study of [NiFe]-hydrogenase maturation

• Applicant: Dr. Basem Soboh
• Subject Area: Biochemistry ; Biophysics ; Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314406875

Understanding the biogenesis of the NiFe cofactor at the molecular level is beneficial for the design of biocatalysts focused on H2 generation. We developed an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase exclusively using purified components. The maturation process included assembly and insertion of the NiFe(CN)2CO cofactor into the large subunit, cleavage of the C-terminal extension, and dimerization with the small subunit to yield active enzyme. Taking advantage of the in vitro system, we were able to probe the functional role of the C-terminal extension of apo-protein during hydrogenase maturation. Our results indicate that the C-terminal extension orchestrates the sequence of events by directing and binding maturation proteins and ensuring that only the cofactor-containing large subunit can continue on the assembly line toward active [NiFe]-hydrogenase. To analyze the composition of protein complexes along the path of NiFe-cofactor biosynthesis, several functional intermediates were isolated at distinct maturation steps from the in vitro assays. Moreover, a supramolecular complex including five maturation proteins together with the large hydrogenase subunit could be reconstituted in vitro. Therefore, one of the major aims of the current proposal is to determine the structure of this highly interesting snapshot and to solve the conformational dynamics of the [NiFe]-maturation machinery using single particle cryo EM. Another major aim of this proposal is to follow the stepwise synthesis and assembly of the cofactor and to analyze the metal center of hydrogenase-intermediates using FTIR and X-ray absorption spectroscopy (XAS). For this purpose, different variants of large subunit precursors have been isolated from defined hydrogenase mutants as well as from in vitro maturation assays. Preliminary XAS studies have been initiated. We were able to provide the first conclusive IR characterization of the in vitro reconstituted “O2-sensitive” [NiFe]-hydrogenase-2. Combining this expertise with our recently developed in vitro maturation system of “O2-tolerant” [NiFe]-hydrogenase-1, we will explore the differences between both enzymes, in particular with respect to O2 and CO inhibition. Investigating “O2-tolerant” and “O2-sensitive” [NiFe]-hydrogenases from the same organism will facilitate a direct comparison and may provide valuable inspiration for the design of O2-tolerant catalysts. An enzyme complex that converts CO and H2O to CO2 and H2., has been previously isolated and biochemically characterized by the applicant. The enzyme complex is formed by a CO-DH and a membrane bound [NiFe] hydrogenase. We aim to determine the structure of the enzyme complex by cryo EM. The protonation/ reduction dynamics in the conversion of CO to CO2 will be follow by in situ ATR FTIR. Protein film electrochemistry will be applied to monitor activity of the enzyme complex by recording catalytic currents spectroscopy.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1927: Iron-Sulfur for Life