A safety cap protects hydrogenase from oxygen attack

Abstract

[FeFe]-hydrogenases are efficient H₂-catalysts, yet upon contact with dioxygen their catalytic cofactor (H-cluster) is irreversibly inactivated. Here, we combine X-ray crystallography, rational protein design, direct electrochemistry, and Fourier-transform infrared spectroscopy to describe a protein morphing mechanism that controls the reversible transition between the catalytic H_ox-state and the inactive but oxygen-resistant H_inact-state in [FeFe]-hydrogenase CbA5H of Clostridium beijerinckii. The X-ray structure of air-exposed CbA5H reveals that a conserved cysteine residue in the local environment of the active site (H-cluster) directly coordinates the substrate-binding site, providing a safety cap that prevents O₂-binding and consequently, cofactor degradation. This protection mechanism depends on three non-conserved amino acids situated approximately 13 Å away from the H-cluster, demonstrating that the 1st coordination sphere chemistry of the H-cluster can be remote-controlled by distant residues.

Fig. 1. The unusual O₂-resistance of [FeFe]-hydrogenase CbA5H.

The H-cluster of CpI and other [FeFe]-hydrogenases is irreversibly destroyed when exposed to O₂. CbA5H is a rare exception, as it reversibly converted into an inactive but O₂-protected state (H_inact), even in the absence of exogenous sulfide. From the O₂-protected state, CbA5H can be reactivated by reduction under anaerobic conditions. ROS: reactive O₂-species resulting from O₂-activation after Fe_d-binding. Green bar: unknown feature or mechanism, protecting the H-cluster of CbA5H from O₂-attack in H_inact.

Fig. 2. X-ray structure of CbA5H^WT crystallized under aerobic conditions (CbA5H^air).

a Cartoon structure of CbA5H^air (PDB ID: 6TTL, chain A; see Supplementary Figure 2 for the homodimer). H- and F-domain are colored in yellow, the N-terminal SLBB domain (soluble ligand binding β-grasp) is presented in green. b Anomalous electron density map, depicting the positions and distances between iron-sulfur clusters, including the H-cluster (stick model), the two F-clusters (FS4A and FS4B) and the additional N-terminal cluster within the SLBB domain (FS4*) located more than 20 Å away from any other cluster. c Potential cluster coordination site within the SLBB domain, consisting of 3 cysteine and one histidine ligand. d, e CbA5H^air exhibits a characteristic structural displacement of the peptide loop containing C367 (TSC-loop) nearby the [2Fe]_H-cluster. d The omit map (Fo-Fc) was contoured at 2 σ (see Supplementary Fig. 17 for a more detailed structural comparison between the loop regions of CpI and CbA5H^air). e Structural alignment, depicting conformational differences between CbA5H^air (yellow) and CpI (white). Panels d and e focus on the [2Fe]_H-cluster and side chains of amino acids which influence anaerobic inactivation, O₂-resistance, and H_inact formation. f Selected parts of an amino acid sequence alignment of CbA5H^WT and CpI, including the polypeptide positions that influence TSC-loop reconfiguration and H_inact state formation in CbA5H^WT.

Fig. 3. Electrochemical and spectroscopic features of CbA5H^WT and mutagenesis variants.

a Comparison of cyclic voltammograms of CbA5H^WT(Cb-WT), CpI (CpI-WT), and Cb-variant C367D (T = 5 °C, pH 7, 1 atm. of H₂, scan rate 3 mV/s, electrode rotation rate 3000 rpm, currents normalized at E = −0.56 V). b Potential step chronoamperometry of CbA5H^WT and C367D (5 °C, pH 7, 1 atm. of H₂, 1000 rpm). P1: H₂-production current at −0.8 V prior to O₂-exposure; P2: potential step to 0 V; P3: injection of 50 μM O₂; P4: five-fold buffer exchange to re-establish anaerobic conditions; P5: potential step to −0.8 V to measure the residual H₂-production current. c ATR-FTIR-spectroscopy of CbA5H^WT and variant C367D prior and after O₂-exposure (pH 8). d Cyclic voltammograms of CbA5H^WT (black) and mutagenesis variants L364F, P386L, A561F, and L364F-A561F (5 °C, pH 7, 1 atm. of H₂, 20 mV/s, 3000 rpm, currents normalized at E = −0.32 V except WT, at −0.56 V). e Chronoamperometry as in panel B, with CbA5H^WT and the same variants as in d. f ATR-FTIR-spectroscopy (pH 8) of the loop variants, prior, and after O₂-exposure. Electrochemical and IR spectroscopic experiments have been repeated for each protein at 3-4 times with consistent results. Source data are provided in a source data file.

Fig. 4. Anaerobic conversion between active and inactive CbA5H^WT monitored by PFE.

a Chronoamperograms recorded to analyze the kinetics of anaerobic (in)activation of CbA5H^WT by stepping between −0.31 and −0.21 V. The boxes along the abscissa depict the sequence of applied potential steps and the corresponding current response (black line). The plot also shows the best fits of models that consider the interconversion between two (green dotted line) or three (red dotted line) species. Experimental parameters: 5 °C, pH 7, 3000 rpm. b Dependence of the rate constants of the “AAI” model (A1 ↔ A2 ↔ H_inact) on potential and pH, based on the analyses of chronoamperograms recorded for CbA5H^WT at pH 10 (squares), 8.5 (triangle), and 7 (circle). Only k_react significantly depends on pH and electrode potential. Source data are provided in a source data file.

Fig. 5. Mechanism for the reversible formation of the O₂-resistant H_inact state in CbA5H^WT.

a, b Structural alignment of CpI (state A₁, blue) and CbA5H^air (H_inact; red) focusing on the TSC-loop. Residues labeled in red determine the structural rearrangement from A₁ to H_inact. Corresponding CpI positions are shown in blue (yellow glow indicates clashes). c Illustration of the AAI mechanism. Main steps (1–3) in the transition from A₁ (blue) to H_inact (red) via A₂ (blue-red) are marked in green, corresponding to structural rearrangements (1-3) shown in a-b. 1: Partial shift of the TSC-loop, including T365, initiating the A₁ to A₂ transition. 2: W371-translocation and α-helix uplift, dragging along C367 closer to Fe_d (green arrows) 3: Binding of C367 to Fe_d (dotted green line) and oxidation of the H-cluster yields the inactive but O₂-resistant H_inact state. Arrow and font sizes reflect relative rates of k₁ and k_-1 or k_inact and k_react, in CbA5H, which define the dynamic equilibrium between A₁, A₂, and H_inact. Under reductive conditions (+e⁻/H⁺) the k_inact/k_react ratio favors A₂, whereas H_inact accumulates under oxidative conditions (−e⁻/H⁺).