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

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 Fd^2--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.

Figure 1

Purification of Ech2.A, to purify a tagged version of Ech2 the plasmid pMU_ech2C-His was transformed in T. kivui. The expression of ech2C-His was under control of the constitutively expressed S-layer promoter (P_slp). The in trans produced Ech2C-His assembled into the Ech2 complex encoded by the genome. The genetically modified Ech2 complex was purified via affinity chromatography. Purified Ech2 was separated by SDS- (B) or SDS-free PAGE (C) and stained with Coomassie Brilliant Blue G250. Ten micrograms of protein was applied to each lane. C, hydrogenase activity of Ech2 was determined with triphenyltetrazolium chloride and methylviologen under an atmosphere of 3% hydrogen. M1, prestained page ruler; M2, high-molecular-weight calibration ruler.

Figure 2

Ech2 activity is strongly inhibited by DCCD.A, H₂ production was measured in an assay mixture containing buffer E and 15 μg Ech2 in the presence of Fd (■), Fd + 100 μM DCCD (▼), or in the absence of Fd (●) or Ech2 (♦) as described in Experimental procedures. B, for inhibition studies 15 μg desalted Ech2 (final Na⁺ concentration in the assay: ≈180 μM) was preincubated in buffer H for 20 min at room temperature in the presence (▲) or absence (■) of 50 mM NaCl with 0 to 500 μM DCCD, respectively. C, inhibition of DCCD at pH 6 (▲) compared with pH 7.5 (■) was tested in buffer I or H. Fd^2-:H⁺ oxidoreductase activity was determined as described in Experimental procedures. H₂ was measured via gas chromatography as described previously (17). All data points are mean ± SEM; N = 3 independent experiments.

Figure 3

Ech2 activity establishes a pH gradient across the membrane.A, H₂ production was measured in an assay mixture containing buffer D in the presence of 240 μg proteoliposomes and Fd (●) or in the absence of Fd (▼) or proteoliposomes (♦) as described in Experimental procedures. H₂ was measured via gas chromatography as described previously (17). B, for inhibition studies 100 to 250 μg proteoliposomes were preincubated with 30 μM TCS, ETH 2120, or 1% [v/v] ethanol for 20 min at room temperature in buffer D (including 10 mM NaCl) before the Fd^2-:H⁺ oxidoreductase reaction was started. C, the generation of a ΔpNa⁺ was measured in an assay mixture containing buffer D, 950 μg proteoliposomes preincubated with 30 μM TCS (▼) or ETH 2120 (♦), in the presence (■) or absence of Fd (▲) as described in Experimental procedures. The Fd^2-:H⁺ oxidoreductase reaction was started with the addition of 10 mM pyruvate as indicated. Radioactivity was determined as described previously (61). D, the generation of a ΔpH was recorded by measuring the fluorescence (excitation: 410 nm, emission: 490 nm) of the pH indicator ACMA as described in Experimental procedures. To induce the establishment of a ΔpH, the assay contained buffer D and 240 (black) or 120 μg (orange) proteoliposomes and the Fd^2-:H⁺ oxidoreductase reaction was started with the addition of 10 mM pyruvate as indicated. To dissipate the electrical field 30 μM TCS was added as indicated. In control assays Fd was omitted (purple), liposomes without reconstituted Ech2 (blue) were used, or proteoliposomes were additionally preincubated with 1% [v/v] ethanol (green), 50 μM DCCD (gray), 30 μM ETH2120 and 10 mM NaCl (brown), 30 μM ETH2120 without the addition of NaCl (pink) or 30 μM TCS (red), respectively. All data points are mean ± SEM; N = 3 independent experiments.

Figure 4

Ech2-catalyzed H⁺transport is electrogenic. The generation of a membrane potential was recorded by measuring the difference in absorption changes (625–587 nm) of oxonol VI and the reduction of Fd (430 nm) simultaneously. The measurements were performed as described in Experimental procedures. A, to induce the establishment of an electrical field, the assay containing 200 μg proteoliposomes was started with 10 mM pyruvate. To dissipate the electrical field 30 μM TCS was added as indicated. B, control assays omitted Fd. N = 3 independent experiments.

Figure 5

The respiratory chain of the thermophilic acetogen T. kivui. Schematic representation of the Ech2 complex and the ATP synthase of the respiratory chain in T. kivui. Exergonic electron transfer from reduced Fd to 2 H⁺ leads to the translocation of H⁺ across the cytoplasmic membrane and the electrochemical H⁺ potential is then the driving force for ATP synthesis. Membrane-bound subunits of Ech2 are colored blue. The electron pathway from the donor to the acceptor is unknown. Cubes, [4Fe-4S] clusters; [Ni-Fe], hydrogenase active site.