Glutamate Gated Proton-Coupled Electron Transfer Activity of a [NiFe]-Hydrogenase

Abstract

[NiFe] hydrogenases are metalloenzymes that catalyze the reversible oxidation of H. While electron transfer to and from the active site is understood to occur through iron-sulfur clusters, the mechanism of proton transfer is still debated. Two mechanisms for proton exchange with the active site have been proposed that involve distinct and conserved ionizable amino acid residues, one a glutamate, and the other an arginine. To examine the potential role of the conserved glutamate on active site acid-base chemistry, we mutated the putative proton donor E to Q in the soluble hydrogenase I from Pyrococcus furiosus using site directed mutagenesis. FTIR spectroscopy, sensitive to the CO and CN ligands of the active site, reveals catalytically active species generated upon reduction with H, including absorption features consistent with the Ni-C intermediate. Time-resolved IR spectroscopy, which probes active site dynamics after hydride photolysis from Ni-C, indicates the EQ mutation does not interfere with the hydride photolysis process generating known intermediates Ni-I and Ni-I. Strikingly, the EQ mutation disrupts obligatory proton-coupled electron transfer from the Ni-I state, thereby preventing formation of Ni-S. These results directly establish E as a proton donor/acceptor in the Ni-S ↔ Ni-C equilibrium.

Proton Transfer in the Catalytic Cycle of [NiFe] Hydrogenases: Insight from Vibrational Spectroscopy

“…96 A conserved glutamate residue (E28 in Hyd-1 notation) located close to the Ni-bound terminal cysteine thiolates is suggested to be important for proton transfer beyond the primary coordination sphere of the active site on the basis of mutagenesis studies, 12 and is the active-site terminus of a number of putative proton-transfer pathways. 11−14,30 Time-resolved photolysis measurements by Dyer and co-workers have established this residue as a proton acceptor site during the Ni a -L to Ni a -SI transition in P. furiosus SH1, 61 consistent with their earlier study which showed that proton transfer is mediated by an amino acid residue with p K a ∼ 7. 42 …”

“…They calculate a Δ G value of −1 kJ/mol for the concerted process on the basis of the relative concentrations of Ni a -L and Ni a -SI during a defined time window following photolysis of Ni a -C, and they make the assumption that Δ G > 0 for oxidation of (b) without coupled deprotonation. 61 While entirely consistent with available data for P. furiosus SH1, this model is not necessarily consistent with the work of Hirota and co-workers on D. vulgaris MF hydrogenase, 110 who showed that the relative populations of protonated (b) and deprotonated (c or d) Ni a -L states are pH-dependent with a calculated Δ G value of −1.2 ± 0.9 kJ/mol for their interconversion. Neither is it consistent with the observation that Ni a -L species observed in Group 1 O 2 -tolerant hydrogenases, even in the dark and at ambient temperature, tend to be biased toward lower wavenumber species and are likely to have a deprotonated active site (vide infra, Figure 10A).…”

“…35 More transient states have also been probed in time-resolved infrared spectroscopic techniques in which transitions between states of a hydrogenase have been triggered by the release of caged electrons or by photolysis of light-sensitive states of the enzyme. 41,42,60 Attention has also focused on the role of conserved amino acids in the region around the active site in controlling proton transfer, 12,54,61−63 and the correlation between the redox state of the proximal cluster and that of the active site. 36,60 These studies have been aided by the availability of site-directed variants of [NiFe] hydrogenases with particular amino acid exchanges in the region of the active site and the proximal iron sulfur cluster.…”

“…36,60 These studies have been aided by the availability of site-directed variants of [NiFe] hydrogenases with particular amino acid exchanges in the region of the active site and the proximal iron sulfur cluster. 12,54,61,63 However, one limitation to recent studies of [NiFe] hydrogenases has been that each vibrational spectroscopy approach has only been applied to one or two different hydrogenases, and it remains to be seen whether the findings from each approach represent aspects of a common [NiFe] hydrogenase mechanism or whether certain details are specific to individual hydrogenases.…”

“…45 The NADP + -reducing soluble hydrogenase (SH) 1 from Pyrococcus furiosus , a hyperthermophilic archaeon, has been studied more extensively using time-resolved IR methods. 41,42,61 …”