Susceptibility of the Formate Hydrogenlyase Reaction to the Protonophore CCCP Depends on the Total Hydrogenase Composition

Janik Telleria Marloth,Constanze Pinske

doi:10.3390/inorganics8060038

Janik Telleria Marloth, Constanze Pinske

Open Access

https://doi.org/10.3390/inorganics8060038

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Journal: Inorganics	Publication Date: May 28, 2020
Citations: 3	License type: CC BY 4.0

Affiliation: Martin Luther University Halle-Wittenberg

Abstract

Fermentative hydrogen production by enterobacteria derives from the activity of the formate hydrogenlyase (FHL) complex, which couples formate oxidation to H2 production. The molybdenum-containing formate dehydrogenase and type-4 [NiFe]-hydrogenase together with three iron-sulfur proteins form the soluble domain, which is attached to the membrane by two integral membrane subunits. The FHL complex is phylogenetically related to respiratory complex I, and it is suspected that it has a role in energy conservation similar to the proton-pumping activity of complex I. We monitored the H2-producing activity of FHL in the presence of different concentrations of the protonophore CCCP. We found an inhibition with an apparent EC50 of 31 µM CCCP in the presence of glucose, a higher tolerance towards CCCP when only the oxidizing hydrogenase Hyd-1 was present, but a higher sensitivity when only Hyd-2 was present. The presence of 200 mM monovalent cations reduced the FHL activity by more than 20%. The Na+/H+ antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) combined with CCCP completely inhibited H2 production. These results indicate a coupling not only between Na+ transport activity and H2 production activity, but also between the FHL reaction, proton import and cation export.

Highlights

Anaerobic or fermentative growth in the absence of oxygen requires that conservation of energy is at its utmost efficiency
carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) Inhibits H2 Production to Different Degrees Depending on the Hydrogenase Composition
Formate is the direct substrate for the formate hydrogenlyase (FHL) complex, but its import via the formate channel FocA could mask the effects seen

Summary

Introduction

Anaerobic or fermentative growth in the absence of oxygen requires that conservation of energy is at its utmost efficiency. The reaction of the formate hydrogenlyase (FHL) complex contributes indirectly to the generation of a proton gradient at the cytoplasmic membrane by consumption of protons (H+ ) from the cytoplasm, diffusion of H2 -gas across the membrane and subsequent oxidation by the two periplasmic H2 -oxidizing hydrogenases (Hyd-1 and Hyd-2) [1] (Figure 1). The hyaB and hybC genes encode the catalytic subunits of Hyd-1 and Hyd-2, respectively [3]. This kind of intracellular syntrophy was initially described for Desulfovibrio species [4]. The two active sites of the FHL complex comprise the [NiFe]-hydrogenase 3 (HycE protein) and the molybdenum- and selenium-containing formate dehydrogenase H (FdhH protein), which function together with three electron-transferring iron-sulfur carrying subunits located in the cytoplasm. Membrane attachment to the membrane-integral HycC and HycD subunits is required for Inorganics 2020, 8, 38; doi:10.3390/inorganics8060038 www.mdpi.com/journal/inorganics

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