Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane‐bound enzyme capable of fixing carbon dioxide to organic acid

Abstract

During mixed‐acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO 2 and H2. Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO 2 to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function.

Hydrogen or formate: Alternative key players in methanogenic degradation

“…Depending on whether or CO 2 was used for calculation, reaction enthalpies ranged from −8.1 to −16.3 kJ per mol, indicating that this part of the reaction energy was not exploited (Table ). Other authors demonstrated hydrogen production from formate with purified formate hydrogenlyase of E. coli (Pinske and Sargent, ). Here, 15.7 mM formate was added to the purified enzyme in the reaction chamber of a hydrogen‐sensing Clark electrode and maximal production of around 600 nmol hydrogen in 1.7 ml volume was observed, with a final formate concentration of 14.9 mM.…”

“…Here, 15.7 mM formate was added to the purified enzyme in the reaction chamber of a hydrogen‐sensing Clark electrode and maximal production of around 600 nmol hydrogen in 1.7 ml volume was observed, with a final formate concentration of 14.9 mM. The final concentrations of formate, hydrogen, and CO 2 (calculated from the consumed formate) were 14.9 mM, 353 µM and 840 µM respectively (Pinske and Sargent, ). From these values and the standard Gibbs’ free reaction energy of −3.4 kJ per mol (Eq. )…”

“…The enzyme has probably not evolved to exploit the complete reaction energy as it solely serves as a pH‐regulating enzyme at high formate concentrations. Although the enzyme is membrane‐associated there is no indication that formate oxidation is coupled to proton translocation across the membrane, and hence it probably does not conserve energy (Pinske and Sargent, ), different from the formate‐oxidizing system of Thermococcus onnurineus (Lim et al ., ; see below).…”

“…Similar enzymes in Syntrophomonas wolfei or Pelobacter acetylenicus may act in a similar manner. On the other hand, production of formic acid by E. coli during mixed acid fermentation leads to a pH decrease of the growth medium, especially when the culture reaches stationary phase (Pinske and Sargent, ). E. coli can compensate this effect by expressing formate hydrogen lyase which oxidizes formate and transfers electrons to protons to form hydrogen as a final product (Pinske and Sargent, ).…”

Membrane Potential as a Regulation Mechanism of Periplasmic Nitrite Reductase Activity: A Mathematical Model

“…Эта возможность просматривается в структуре HycD/HycC субъединиц Hyd-3 фермента, обладающих значительным сходством с респираторным комплексом НАДН:хиноноксидоредуктазы (Complex 1) [27,16,28], и продемонстрирована в экспериментах у двойных мутантов c делециями оперонов hya [Hyd-1] и hyb [13], проведенных при рН = 6.5 в ферментирующих условиях, что совпадает с условиями роста клеток в проточном хемостате [20] в отсутствие нитрита. В случае прямой транслокации протонов через FHL-1 комплекс, механизм формирования мембранного потенциала будет идентичен таковому с участием FHL-2 комплекса, который рассматривается далее.…”