Spatiotemporal Assembly and Function of the Modular Formate Hydrogenlyase Complex

• Applicant: Dr. Constanze Pinske
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Biochemistry
• Term: from 2016 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 310984203

When Escherichia coli is grown under fermentative conditions, it produces H2 from the disproportionation of formate and the enzyme responsible is the remarkable seven subunit formate hydrogenlyase (FHL) complex. FHL is a membrane bound enzyme that shares structural features with the respiratory NADH dehydrogenase Complex I. FHL comprises a cytoplasmic domain including a molybdenum dependent formate dehydrogenase (formate DH) where formate is oxidized to CO2 and electrons, a [NiFe] hydrogenase that uses those electrons to produce H2, and three iron sulfur containing electron transfer proteins that wire the two catalytic domains. In addition, the FHL cytoplasmic domain is attached to a membrane domain comprising two subunits termed HycC and HycD. Although, significant advances have been made recently in purification of the entire complex, or stable subcomplexes therefrom, as well as native PAGE activity characterization (1, 2) key points of the protein specific maturation and assembly of FHL proteins are not yet understood. In particular, the role of the non structural, metal binding chaperone HycH, that has an impact on FHL activity during assembly, is unknown. Another question poses the crosstalk between the hydrogenase, the formate DH and the various [FeS] cluster containing electron transfer subunits, as well as its implication on FHL function.As part of the hydrogen producing machinery, the FHL complex is a promising candidate for biotechnological H2 production and additionally our very recent work also showed its ability to be employed in CO2 fixation. Understanding the interplay of the various metal containing subunits, their specific needs in maturation and finding the optimal time point for assembly will help in engineering of a more robust catalytic complex. Thereby, the FHL complex could potentially outcompete biotechnologically established H2 generating systems because of its ease to use. Therefore, the aims of this proposal are to characterise the post cofactor insertion FHL complex assembly biochemically and to clarify the mechanistic involvement of its specific maturation proteins.

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