Conserved Histidine Adjacent to the Proximal Cluster Tunes the Anaerobic Reductive Activation of Membrane-Bound [NiFe] Hydrogenase-1.

[NiFe] hydrogenases are electrocatalysts that oxidize H at a rapid rate without the need for precious metals. All membrane-bound [NiFe] hydrogenases (MBH) possess a histidine residue that points to the electron-transfer iron sulfur cluster closest ("proximal") to the [NiFe] H-binding active site. Replacement of this amino acid with alanine induces O sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O-induced over-oxidation of the [FeSCys] proximal cluster possessed by all O-tolerant MBH. We have created an Hyd-1 His-to-Ala variant and report O-free electrochemical measurements at high potential that indicate the histidine-mediated [FeSCys] cluster-opening/closing mechanism also underpins anaerobic reactivation. We validate these experiments by comparing them to the impact of an analogous His-to-Ala replacement in Hyd-2, a [NiFe]-MBH that contains a [FeS] center.