Enzymatic and spectroscopic properties of a thermostable [NiFe]‑hydrogenase performing H2-driven NAD+-reduction in the presence of O2

Abstract

Biocatalysts that mediate the H₂-dependent reduction of NAD⁺ to NADH are attractive from both a fundamental and applied perspective. Here we present the first biochemical and spectroscopic characterization of an NAD⁺-reducing [NiFe]‑hydrogenase that sustains catalytic activity at high temperatures and in the presence of O₂, which usually acts as an inhibitor. We isolated and sequenced the four structural genes, hoxFUYH, encoding the soluble NAD⁺-reducing [NiFe]‑hydrogenase (SH) from the thermophilic betaproteobacterium, Hydrogenophilus thermoluteolus TH-1^T (Ht). The HtSH was recombinantly overproduced in a hydrogenase-free mutant of the well-studied, H₂-oxidizing betaproteobacterium Ralstonia eutropha H16 (Re). The enzyme was purified and characterized with various biochemical and spectroscopic techniques. Highest H₂-mediated NAD⁺ reduction activity was observed at 80°C and pH6.5, and catalytic activity was found to be sustained at low O₂ concentrations. Infrared spectroscopic analyses revealed a spectral pattern for as-isolated HtSH that is remarkably different from those of the closely related ReSH and other [NiFe]‑hydrogenases. This indicates an unusual configuration of the oxidized catalytic center in HtSH. Complementary electron paramagnetic resonance spectroscopic analyses revealed spectral signatures similar to related NAD⁺-reducing [NiFe]‑hydrogenases. This study lays the groundwork for structural and functional analyses of the HtSH as well as application of this enzyme for H₂-driven cofactor recycling under oxic conditions at elevated temperatures.

Keywords: Biotechnology; Cofactor recycling; Electron paramagnetic resonance spectroscopy; Enzyme kinetics; Flavin; Hydrogen; Hydrogenase; Infrared vibrational spectroscopy; Iron; Iron‑sulfur cluster; Nickel; Nuclear resonance vibrational spectroscopy; Oxyhydrogen reaction; Pyridine nucleotide; Respiratory Complex I.

Fig. 1.

Arrangement of the HtSH-related genes (a), proposed subunit/cofactor composition (b), and observed active site redox states of HtSH (c). Genes hoxF, U, Y, and H encode the subunits of the SH protein, while hoxA has presumably a regulatory function. Upon insertion of the [NiFe] active site, the hoxW gene product mediates cleavage of a C-terminal extension of the HoxH subunit. The proposed cofactor composition in b is derived from amino acid sequence comparisons with the corresponding subunits of ReSH and Complex I from Thermus thermophilus (see Fig. S1) and analogies to the well-characterized ReSH. The assignment of active site species and their interconversions shown in c is based on IR and EPR spectroscopic analyses (see below). Redox states highlighted in green belong to the catalytic conversion of H₂, while the orange ones represent inactive states that – except for Ni_r-S – require reductive treatment to be converted into the Ni_a-S state. The unassigned oxidized state labelled with n/a is unprecedented (see below).

Fig. 2.

Purification of the HtSH protein. A protein amount of 30 μg of soluble extract (SE) and 5 μg of HtSH purified by affinity chromatography (AC) and selected fractions (from the subsequent size exclusion chromatography (SEC) were electrophoretically separated on a 12 % SDS-polyacrylamide gel and subsequently stained with Coomassie brilliant blue. The specific H₂-driven NAD⁺ reduction activity (U mg⁻¹ of protein) of each fraction is specified below. Lane M contains marker proteins and their corresponding molecular weights are given on the left hand side.

Fig. 3.

Dependence of H₂-driven NAD⁺ reduction activity of purified HtSH protein on the addition of reductants TCEP and NADH. The assay was performed at 50 °C in 50 mM bis-Tris, pH 6.5, supplemented with 1 mM NAD⁺, 0.5 mM NiCl₂, 5 mM MgSO₄, 2 μM FMN, and varying amounts of TCEP, NADH and HtSH. The lag time refers to the time elapsed from assay start until full activity was achieved. 100 % activity refers to 19 U mg⁻¹ of protein.

Fig. 4.

Activity of purified HtSH protein at different pH values. The graph depicts the H₂-dependent NAD⁺ reduction activities of HtSH (grey bars) as well as the H₂:benzyl viologen (orange symbols) and NADH:benzyl viologen (blue symbols) oxidoreductase activities of the individual HtSH modules. The measurements were performed as described in materials and methods with 45 nM of HtSH in an universal buffer composed of 16 mM citrate, 16 mM Tris, and 16 mM glycine. Activities were measured at a temperature of 50 °C in the presence of either of 1 mM NAD⁺, 1mM NADH, or 5 mM benzyl viologen, in addition to 0.5 mM NiCl₂, 5 mM MgSO₄, 2 μM FMN, and 0.75 mM TCEP.

Fig. 5.

Temperature dependence of the H₂-dependent NAD⁺ reduction activity of purified HtSH protein. The measurements were performed as described in materials and methods with 45 nM of HtSH in 50 mM bis-Tris buffer, pH 6.5, containing 1 mM NAD⁺, 0.5 mM NiCl₂, 5 mM MgSO₄, 2 μM FMN, and 0.75 mM TCEP. If the error bars are not visible, they are equal or smaller than the symbol size.

Fig. 6.

IR (left) and EPR (right) spectra of HtSH recorded under different redox conditions. Samples were prepared as described in materials and methods and measured in the as-isolated, oxidized state (black spectra) or in their reduced states (red spectra: samples reduced with TCEP and NADH; blue spectra: samples reduced with TCEP, NADH, and H₂). IR spectra were acquired at 10 °C, while EPR spectra were recorded at either 10 K (d) or 35 K (e, f).