The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• The formate hydrogenlyase (FHL) complex in Escherichia coli is essential for hydrogen production.
• FHL is a multi-protein complex primarily encoded by the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3).
• The HycH protein's role in FHL assembly was previously unclear, with a predicted accessory function.

Purpose of the Study:

• To investigate the function of the HycH protein in the assembly and maturation of the FHL complex.
• To identify specific residues in HycH involved in its interaction with FHL subunits and its activity.
• To explore potential overlapping functions of HycH with other hydrogenase maturation machinery.

Main Methods:

• Characterization of a HycH-deficient mutant strain to assess FHL activity and subunit stability.
• Analysis of HycH interactions with unprocessed HycE subunits using variant proteins.
• Generation and testing of HycH variants to determine the role of specific residues in interaction and activity.

Main Results:

• HycH is essential for FHL activity, stabilizing electron transfer subunits and interacting with unprocessed HycE.
• Specific HycH residues (R70/71/K72, Y79, E81, Y128) are involved in HycE interaction but not essential for FHL activity.
• HycH H37 variants negatively impacted FHL activity, while Y30 variants were unstable.
• HycH shares overlapping function with HyfJ, indicating shared maturation machinery between Hyd-3 and Hyd-4.

Conclusions:

• HycH functions as a novel dual-role assembly chaperone for cytoplasmic [NiFe]-hydrogenase.
• This study reveals the first instance of shared maturation machinery components between Hyd-3 and Hyd-4 complexes in E. coli.
• HycH's role extends beyond a simple accessory function, being critical for FHL complex stability and assembly.