Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases

Abstract

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H₂ their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H₂O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

Fig. 1

Putative PT pathway in CpI and H₂ evolution activities of SDM variants. a The PT pathway of CpI (PDB: 4XDC) is presented as stick structure with individually colored residues while the [4Fe-4S]-cluster and water molecules are shown as spheres. The substitutions applied in this study for individual positions are shown in parentheses below the respective position labels. b H₂-production activity of SDM variants targeting the putative PT pathway in CpI and HydA1. H₂ production activities of PT pathway variants determined at pH 6.8 are presented in % relative to the respective wild-type activity. Activity bars for different variants of the same position exhibit corresponding basic colors but different shades. Relative activities higher than 5% are shown in the upper part of the discontinuous scale. Wild-type CpI and HydA1 exhibit activities of 2576 ± 107 and 862 ± 46.5 µmol H₂ per mg per min, respectively. The bars represent mean values from at least three independent measurements, including standard deviations. Details on the in vitro assay are presented in Supplementary Fig. 1

Fig. 2

pH-activity profiles of wt-CpI and wt-HydA1 compared to selected SDM variants. a HydA1 and E144A; b wt-CpI and variants of position E282; c wt-CpI and variants of position E279; d wt-CpI and selected variants of positions R286, S319, and C299. H₂ production activities were determined for different buffers, covering a pH gradient between 5 and 9 in steps of 0.5 pH units. Relative values correspond to % of maximum activity obtained throughout the entire pH gradient. Black plots indicate the relative pH-dependent activities of CpI and HydA1 wild-type enzymes. All values are mean values ± standard deviations from at least three independent measurements

Fig. 3

Infrared spectra of wild types and PT pathway variants. The frequency regime of the H-clusters’ CO ligands is shown (2000–1785 cm⁻¹). For ATR-FTIR spectroscopy, the buffer was set to pH 8 and the rehydrated samples were purged with 100% H₂ for 5 min. a, c Auto-oxidation in absence of H₂ (i.e. purged with N₂) was exploited to likewise enrich for all examined proteins the oxidized resting state, H_ox (gray bands). Some CpI variants tend to accumulate H_oxH in parallel with H_ox (e.g. C299A bands at frequencies 1975/1953/1809). b, d When shifting from N₂ to H₂ the spectrum of wild-type protein changes to different fractions of reduced species including H_red (cyan) and H_sred (red) as well as H_red´ (magenta). Most PT pathway variants accumulate H_hyd (blue) instead or in addition to a mix of reduced states. For precise state-specific vibrational signals of CpI and HydA1 see Supplementary Table 7

Fig. 4

Structural features of SDM variants targeting the putative PT pathway in CpI. Structures of nine SDM variants are superimposed as cartoon-loop models together with the 4XDC wild-type structure. No unspecific differences are observed. For each variant an enlargement of its electron density map in the putative PT pathway and the corresponding sticks model has been aligned with the structure of wild-type protein (H₂O molecules and carbon atoms colored in black). For the CpI proteins local structural differences near the site of mutagenesis are depicted (for the HydA1 variants see Supplementary Fig. 5). Simulated annealing omitting maps (F_o − F_c) were contoured at 3σ except for E282D, which was contoured at 1.9σ due to its comparatively low resolution. Chain B provides a more flexible N terminus but a more rigid H-domain where both the PT pathway and the active center are located. Therefore, all the structural information of CpI was derived from chain B if not stated otherwise

Fig. 5

Hydrogen-bonding pattern in the catalytic PT pathway of the H_ox state. H-bond pattern of H_ox according to pKa values of the residues calculated for the structure of wild-type CpI (4XDC) via PROPKA (see red numbers in parentheses). The pKa of the adt-ligand in H_ox is derived from a previous study. The blue numbers indicate the distances between neighboring positions of the PT pathway. The arrow labeled “SB” indicates a presumptive salt bridge contact between R286 and deprotonated E282

Fig. 6

Influence of SDM on the proton transfer mechanism during H₂-uptake in CpI. Effects of SDM on the proposed proton transfer mechanism for selected CpI variants: C299A (a), E279A (b), E282A (c), and E282Q (d). H₂-binding induces a shift in the H-bond pattern (from mode 1 to mode 2) and initiates the catalytic mechanism during which the H-bond pattern repeatedly shifts between modes 1 (blue) and 2 (green) while promoting a stepwise proton release via the PT pathway (for details see Supplementary Fig. 9+14). The pKa values of adt-ligand at different redox states are derived from previous studies^,. The mutated residues and hydrogen atoms from substrate were highlighted as red. The green double arrow indicates a putative salt bridge contact. In c (E282A), the pink shading area indicates a slowed-down but still functioning proton transfer. Protons presented in blue close to the [4Fe]_H sub-cluster originate from the recently described regulatory PT pathway^,,, which is independent of substrate/product transfer