A purified energy-converting hydrogenase from Thermoanaerobacter kivui demonstrates coupled H+-translocation and reduction in vitro

J Biol Chem. 2022 Aug;298(8):102216. doi: 10.1016/j.jbc.2022.102216. Epub 2022 Jun 30.

Abstract

Energy-converting hydrogenases (Ech) are ancient, membrane-bound enzymes that use reduced ferredoxin (Fd) as an electron donor to reduce protons to molecular H2. Experiments with whole cells, membranes and vesicle-fractions suggest that proton reduction is coupled to proton translocation across the cytoplasmatic membrane, but this has never been demonstrated with a purified enzyme. To this end, we produced a His-tagged Ech complex in the thermophilic and anaerobic bacterium Thermoanaerobacter kivui. The enzyme could be purified by affinity chromatography from solubilized membranes with full retention of its eight subunits, as well as full retention of physiological activities, i.e., H2-dependent Fd reduction and Fd2--dependent H2 production. We found the purified enzyme contained 34.2 ± 12.2 mol of iron/mol of protein, in accordance with seven predicted [4Fe-4S]-clusters and one [Ni-Fe]-center. The pH and temperature optima were at 7 to 8 and 66 °C, respectively. Notably, we found that the enzymatic activity was inhibited by N,N'-dicyclohexylcarbodiimide, an agent known to bind ion-translocating glutamates or aspartates buried in the cytoplasmic membrane and thereby inhibiting ion transport. To demonstrate the function of the Ech complex in ion transport, we further established a procedure to incorporate the enzyme complex into liposomes in an active state. We show the enzyme did not require Na+ for activity and did not translocate 22Na+ into the proteoliposomal lumen. In contrast, Ech activity led to the generation of a pH gradient and membrane potential across the proteoliposomal membrane, demonstrating that the Ech complex of T. kivui is a H+-translocating, H+-reducing enzyme.

Keywords: Thermoanaerobacter kivui; acetogenic metabolism; energy-converting hydrogenase (Ech); extremophile; proteoliposomes; proton translocation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • Ferredoxins / metabolism
  • Hydrogenase* / chemistry
  • Oxidation-Reduction
  • Phylogeny
  • Protons
  • RNA, Ribosomal, 16S / metabolism
  • Sequence Analysis, DNA
  • Thermoanaerobacter

Substances

  • Ferredoxins
  • Protons
  • RNA, Ribosomal, 16S
  • Hydrogenase

Supplementary concepts

  • Thermoanaerobacter kivui