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Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli.

ABSTRACT: The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H₂ during fermentation, its reversibility, allowing H₂-dependent CO₂ reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically and anaerobically isolated forms at resolutions reaching 2.6 Å. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We evaluate possible structural determinants of the bias toward hydrogen production over its oxidation and describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in redox-dependent regulation of FdhF interaction with the complex.

SUBMITTER: Steinhilper R

PROVIDER: S-EPMC9474812 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli.

Steinhilper Ralf R Höff Gabriele G Heider Johann J Murphy Bonnie J BJ

Nature communications 20220914 1

The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H2 during fermentation, its reversibility, allowing H2-dependent CO2 reduction, and its evolutionary link to respiratory complex I. FHL has been studied for ...[more]

PMID: 36104349

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Project description:Trabulsiella guamensis is a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterized Escherichia coli formate hydrogenlyase (FHL-2 Ec ) complex. The T. guamensis FHL-2 (FHL-2 Tg ) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2 Tg complex catalyzes the disproportionation of formate to CO2 and H2 FHL-2 Tg has activity similar to that of the E. coli FHL-1 Ec complex in H2 evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2 Tg operon in the heterologous E. coli host has now enabled us to unambiguously prove FHL-2 Tg activity, and it allowed us to characterize the FHL-2 Tg complex biochemically. Although the formate dehydrogenase (FdhH) gene fdhF is not contained in the operon, the FdhH is part of the complex, and FHL-2 Tg activity was dependent on the presence of E. coli FdhH. Also, in contrast to E. coli, T. guamensis can ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2 Tg and the H2-forming FHL-2 Tg under these conditions.IMPORTANCE Biological H2 production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2 production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2 producers, and the prototype FHL-1 Ec complex in E. coli is well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2 Ec complex, allow a first biochemical characterization of T. guamensis's fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

Project description:Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

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Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli.

Publications

Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli.

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