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

Ute Lindenstrauß, Philipp Skorupa, Jennifer S. McDowall, Frank Sargent, Constanze Pinske (Lead / Corresponding author)

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The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterized and found to have significantly reduced FHL activity due to instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81, and the Y128 variants exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity but not the interaction with HycE was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was found to be unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase 4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase.

Original languageEnglish
Pages (from-to)2937-2350
Number of pages14
JournalBiochemical Journal
Issue number17
Early online date18 Jul 2017
Publication statusPublished - Sept 2017


  • Formate Hydrogenlyase
  • Hydrogen
  • HycH
  • [NiFe]-hydrogenase
  • Chaperone
  • Complex assembly


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