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. 2014 Sep 23;111(38):E3948-56.
doi: 10.1073/pnas.1407927111. Epub 2014 Aug 25.

Bacterial Formate Hydrogenlyase Complex

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Free PMC article

Bacterial Formate Hydrogenlyase Complex

Jennifer S McDowall et al. Proc Natl Acad Sci U S A. .
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Abstract

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H2 even at high partial pressures of H2. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Keywords: PFE; bacterial hydrogen metabolism.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The genetics and predicted structure of formate hydrogenlyase. (A) The genetic organization of the E. coli hycABCDEFGHI operon and the fdhF gene. Genes colored gray do not encode components of the final enzyme. The gene encoding the catalytic subunit of Hyd-3 is indicated. (B) Predicted architecture of the E. coli FHL complex. Proteins are color-coded corresponding to their respective genes from A. (C) Cartoon of E. coli Complex I color-coded to highlight similarity with FHL.
Fig. 2.
Fig. 2.
Affinity tagging of formate hydrogenlyase components. (A) The affinity tags do not interfere with FHL activity. The parental strain (MG1655) and derivatives expressing hycBHis (Bhis), hycEHis (Ehis), hycFHis (Fhis), and hycGHis (Ghis) were grown anaerobically in LB media (pH 6.4) supplemented with 0.4% (wt/vol) glucose. Cells were harvested by centrifugation, washed twice in 50 mM Tris⋅HCl (pH 7.4), and intact cells then assayed for formate-dependent H2 evolution in a H2-sensing electrode as described (5, 47). Error bars represent the SEM, n = 3. (B) FHL Fe−S subunits remain membrane bound following cell lysis. Strains were grown under fermentative conditions before being harvested, washed, and fractionated in the absence of detergent. Equal proportions of crude extracts (CE), total membranes (TM), and soluble proteins (S) were separated by SDS-PAGE and challenged with an anti-His monoclonal antibody (Upper). Fractionation and loading control immunoblots toward the HybO integral membrane protein (Middle), and the soluble maltose binding protein (MalE, Lower) are included. (C) FHL can be isolated in a single step. Strains were lysed in a detergent mixture before extracts were applied to IMAC columns and bound proteins eluted and pooled. Approximately 10 μg of concentrated samples were separated by SDS-PAGE and stained with Instant Blue. All protein bands analyzed by tryptic peptide mass spectrometry are labeled: 1, positively identified as FdhF; 2, HycE; 3, HycG; 4, HycB; 5, HycF; and 6, predominantly fragments of HycE. (D) Isolated HycEHis can be identified by Western immunoblotting. A CE of MG059e1 (hycEHis) was prepared from a detergent mixture (6 g of cells in 40 mL solution) and applied to a 5-mL IMAC column. The unbound protein (column flow-through, FT) was collected in 40 mL total. Bound proteins were eluted in a 30-mL gradient of 50–1,000 mM imidazole, and 10 × 1 mL peak fractions were collected, seven of which were applied to 12% (wt/vol) SDS-PAGE. Separated proteins were transferred to nitrocellulose and challenged with an anti-His monoclonal antibody.
Fig. 3.
Fig. 3.
FHL activity of the isolated FHL complex assayed in vitro. FHL was isolated for strain MG059e1 (hycEHis), suspended in 100 mM Mes pH 6.0, and incubated under N2 with 2% CH4 as calibrant gas for gas chromatography. Potassium formate solution (pH 6.5) was added to 50 mM (final concentration), and the reaction was allowed to proceed at 25 °C. H2 concentration in the headspace was determined by gas chromatography. Error bars represent SE where n = 3, except for the data point at 120 min, where n = 2.
Fig. 4.
Fig. 4.
Size-exclusion chromatography of the HycEHis-containing FHL complex. (A) Elution profile of HycEHis associated protein in the presence of detergent. The MG059e1 strain was lysed in a detergent mixture and applied to IMAC columns, and bound proteins were eluted and pooled in buffers containing 0.05% (wt/vol) DDM. Approximately 500 μg of pooled protein was then separated by SEC in buffers containing DDM. Fractions were separated by SDS-PAGE and stained with Instant Blue. (B) The peak fraction containing HycC and HycD is indicated by the asterisk. (C) Elution profile of HycEHis associated proteins in the absence of detergent. The strain was lysed in a detergent mixture and subjected to IMAC in the absence of all detergent. Approximately 500 μg of pooled protein was then separated by SEC in buffers devoid of detergent. Fractions were analyzed by SDS-PAGE (D). Proteins were identified by mass spectrometry and direct sequencing.
Fig. 5.
Fig. 5.
Protein film voltammetry study of Hyd-3. Cyclic voltammograms were carried out at pH 6.0 (37 °C) under different amounts of H2 as indicated. Nitrogen was used as the carrier gas, and the gas flow rate was 300 standard cubic centimeters per min (sccm). The electrode potential was increased from −0.56 to +0.24 V vs. SHE and then reversed, with a scan rate of 1 mV/s. Electrode rotation rate was 1,000 rpm.
Fig. 6.
Fig. 6.
Chronoamperometric determination of (A and B) the product inhibition (KiH2/H+) constant during proton reduction at −0.558 V vs. SHE and (D and E) the Michaelis−Menten (KMH2) constant during H2 oxidation at −0.108 V vs. SHE. Both experiments were carried out at pH 6.0, 37 °C, rotation rate 1,000 rpm, gas flow 1,000 sccm. Film loss was estimated according to the steady-state slope at each H2 concentration, and data were corrected accordingly. (C) A comparison of KiH2 values previously reported for various hydrogenase enzymes. (F) A comparison of KMH2 values previously reported for various hydrogenase enzymes. Conditions for each measurement vary and are detailed in Table S1. ReMBH, Ralstonia eutropha membrane-bound hydrogenase (MBH); RmMBH, R. metallidurans MBH; Ec Hyd1, E. coli Hyd-1 (MBH); DgH, Desulfovibrio gigas hydrogenase; DfH, D. fructosovorans hydrogenase; Ec Hyd-1, E. coli Hyd-2 (MBH); EcHyd3, E. coli Hyd-3; Dmb, Desulfomicrobium baculatum [NiFeSe] hydrogenase; CrHydA1, Chlamydomonas reinhardtii HydA1; CaHydA, Clostridium acetobutylicum HydA.
Fig. 7.
Fig. 7.
Chronoamperometry experiments designed to assess the KiCO for Hyd-3 during H+ reduction (A and B) and H2 oxidation (C and D). Experimental conditions were as follows: pH 6.0, 37 °C, rotation rate 1,000 rpm, gas flow 300 sccm, 100% Ar (A) or 10% H2 in Ar (C), −0.537 V vs. SHE (A), −0.087 V vs. SHE (C). KiCO values were 56 ± 17 µM for H+ reduction (B) and 8.2 ± 1.8 µM for H2 oxidation (D). Film loss was estimated according to the steady-state slope at each CO concentration, and data were corrected accordingly.

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