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 H 2 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 2 ‐binding active site. Replacement of this amino acid with alanine induces O 2 sensitivity, and this has been attributed to the role of the histidine in enabling the reversible O 2 ‐induced over‐oxidation of the [Fe 4 S 3 Cys 2 ] proximal cluster possessed by all O 2 ‐tolerant MBH. We have created an Escherichia coli Hyd‐1 His‐to‐Ala variant and report O 2 ‐free electrochemical measurements at high potential that indicate the histidine‐mediated [Fe 4 S 3 Cys 2 ] 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 [Fe 4 S 4 ] center.