Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase

Abstract

[NiFe]-hydrogenases catalyze the reversible cleavage of H2 into two protons and two electrons at the inorganic heterobimetallic NiFe center of the enzyme. Their catalytic cycle involves at least four intermediates, some of which are still under debate. While the core reaction, including H2/H- binding, takes place at the inorganic cofactor, a major challenge lies in identifying those amino acid residues that contribute to the reactivity and how they stabilize (short-lived) intermediate states. Using cryogenic infrared and electron paramagnetic resonance spectroscopy on the regulatory [NiFe]-hydrogenase from Cupriavidus necator, a model enzyme for the analysis of catalytic intermediates, we deciphered the structural basis of the hitherto elusive Nia-L intermediates. We unveiled the protonation states of a proton-accepting glutamate and a Ni-bound cysteine residue in the Nia-L1, Nia-L2, and the hydride-binding Nia-C intermediates, as well as previously unknown conformational changes of amino acid residues in proximity of the bimetallic active site. As such, this study unravels the complexity of the Nia-L intermediate and reveals the importance of the protein scaffold in fine-tuning proton and electron dynamics in [NiFe]-hydrogenase.

Mechanistic principles of hydrogen evolution in the membrane-bound hydrogenase

“…(31)). The proton transfer to Cys68L (3G1, -4.1 kcal mol -1 , Figure S8) is also exergonic, suggesting that the proton could populate different rotameric configurations of both Cys374L and Cys68L, consistent with the experimentally observed multiple forms of the Ni-L state (46,47). However, in contrast to the energetically favored Glu21L-mediated pathway, we find that the His75L-mediated proton transfer to the cluster is strongly endergonic (Figure S8 (48).…”

“…We note that several structural models for the Ni-L (46,47) and Ni-R (23,56,57) states have been reported based on spectroscopic signatures, and indicating different structural isomers. For instance, temperature-dependent FTIR studies of the soluble [NiFe] hydrogenases observed protonation/deprotonation of the residue homologous to Cys374L (Cys546 in DvMF) with an ΔH and ΔS of 1.5 ± 0.8 kcal mol -1 and 6.1 ± 10.3 kcal mol -1 K -1 , suggesting that the cysteine residue can indeed undergo protonation change in the Ni-L state (46).…”

Replacing a Cysteine Ligand by Selenocysteine in a [NiFe]-Hydrogenase Unlocks Hydrogen Production Activity and Addresses the Role of Concerted Proton-Coupled Electron Transfer in Electrocatalytic Reversibility

“…Investigations of the Ni−C to Ni a −SI interconversion by transient spectroscopy lend strong support for a concerted process, 31,32 and recent studies of the regulatory hydrogenase from Cupriavidus necator by cryo-IR and EPR produced a particularly detailed picture of the proton-transfer steps in that enzyme, elaborating on the pathway shown in Figure 2A. 38 The crucial cysteine both coordinates Ni and serves as a H + mediator. An attractive approach for probing the role of a cysteine-S atom is to exchange the cysteine for a selenocysteine (Sec, one-letter code U) by recombinant methods.…”

“…The Ni−C state is in tautomeric equilibrium with Ni(I) species known collectively as Ni−L: IR spectroscopic studies have revealed that both Ni−L and Ni−R exist in several forms, differing in the location of the H + that has migrated locally without leaving the enzyme. 32,33,35,38 The Ni−L species form upon illumination at low temperature, but their detection under normal catalytic conditions has largely been restricted to O 2 -tolerant [NiFe] hydrogenases, suggesting that the equilibrium otherwise strongly favors Ni−C. 33,38,42,43 During H 2 oxidation, Ni−C is converted to a Ni(II) form known as Ni a − SI.…”

“…32,33,35,38 The Ni−L species form upon illumination at low temperature, but their detection under normal catalytic conditions has largely been restricted to O 2 -tolerant [NiFe] hydrogenases, suggesting that the equilibrium otherwise strongly favors Ni−C. 33,38,42,43 During H 2 oxidation, Ni−C is converted to a Ni(II) form known as Ni a − SI. Investigations of the Ni−C to Ni a −SI interconversion by transient spectroscopy lend strong support for a concerted process, 31,32 and recent studies of the regulatory hydrogenase from Cupriavidus necator by cryo-IR and EPR produced a particularly detailed picture of the proton-transfer steps in that enzyme, elaborating on the pathway shown in Figure 2A.…”