Crystallographic studies of [NiFe]-hydrogenase mutants: towards consensus structures for the elusive unready oxidized states

Abstract

Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.

Structural differences between the active sites of the Ni-A and Ni-B states of the [NiFe] hydrogenase: an approach by quantum chemistry and single crystal ENDOR spectroscopy

“…9,24,27,[29][30][31][32][33][34][35][36][37][38] Most notably Li and Hall in 2001 performed calculations on multiple model structures for the Ni-A state and found that a hydroxide bridge is most likely, albeit with a reduced, protonated thiolate. 24 Recent work by Pardo et al 27 predicted a sulfenated cysteine as well as a hydroperoxo bridging ligand, following previous suggestions by Volbeda et al 28 The latest crystal structure determination by Volbeda et al, 39 however, contains an oxygenated thiolate, S=O and a hydroxide bridge. A misconception is that O 2 is required for formation of Ni-A; Albracht et al have shown that oxidation with molecular oxygen exclusively gives rise to the Ni-B state within 158 ms. 22 Additionally, O 2 is a triplet state, and the presence of an intact O 2 molecule near the active site would completely alter the EPR spectrum.…”

A Threonine Stabilizes the NiC and NiR Catalytic Intermediates of [NiFe]-hydrogenase

“…All variants contain a significant fraction of Cys-75 in the oxidized sulfenate form that has recently been assigned to the unready NiA and NiSU states (41). The different sulfenate conformation in T18V might explain the different NiA EPR signal that is observed for this variant (Fig.…”

“…5A). The signal was recently assigned to a persulfide containing state called Ni-S ox (41 (Fig. 5B).…”

Theoretical Insights into the Unique Ligation of [Fe4S4] Iron–Sulfur Clusters

“…There is more flexibility in the choice of the fourth ligand . For example the most solvent exposed (distal) cubane found in [FeFe]‐hydrogenase from Clostridium pasteurianum and [NiFe]‐hydrogenase from Desulfovibrio fructosovorans features histidine as a fourth ligand (Figure b). Carboxylic group of the aspartate side chain (Figure c) is most probably one of the ligating agents in ferredoxin from Pyrococcus furiosus , and iron–sulfur assembly protein A (IspaA) from Acidithiobacillus ferrooxidans .…”

“…Both are observed in nature, e.g. former in [FeFe]‐hydrogenase from Clostridium pasteurianum and latter in [NiFe]‐hydrogenase from Desulfovibrio fructosovorans . Protonation of these two ligands leads to structures denoted as t‐HisH and p‐HisH.…”