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

[NiFe] hydrogenases are electrocatalysts that oxidize H2 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] H2 -binding active site. Replacement of this amino acid with alanine induces O2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O2 -induced over-oxidation of the [Fe4 S3 Cys2 ] proximal cluster possessed by all O2 -tolerant MBH. We have created an Escherichia coli Hyd-1 His-to-Ala variant and report O2 -free electrochemical measurements at high potential that indicate the histidine-mediated [Fe4 S3 Cys2 ] 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 Escherichia coli Hyd-2, a [NiFe]-MBH that contains a [Fe4 S4 ] center.