Redox tuning of the H-cluster by second coordination sphere amino acids in the sensory [FeFe] hydrogenase from

• Published in: Chemical science (Cambridge) Vol. 14; no. 13; pp. 3682 - 3692
• Main Authors: Chongdar, Nipa, Rodrguez-Maciá, Patricia, Reijerse, Edward J, Lubitz, Wolfgang, Ogata, Hideaki, Birrell, James A
• Format: Journal Article
• Published: 29.03.2023
• ISSN: 2041-6520; 2041-6539
• DOI: 10.1039/d2sc06432d

[FeFe] hydrogenases are exceptionally active catalysts for the interconversion of molecular hydrogen with protons and electrons. Their active site, the H-cluster, is composed of a [4Fe-4S] cluster covalently linked to a unique [2Fe] subcluster. These enzymes have been extensively studied to understand how the protein environment tunes the properties of the Fe ions for efficient catalysis. The sensory [FeFe] hydrogenase (HydS) from Thermotoga maritima has low activity and displays a very positive redox potential for the [2Fe] subcluster compared to that of the highly active prototypical enzymes. Using site directed mutagenesis, we investigate how second coordination sphere interactions of the protein environment with the H-cluster in HydS influence the catalytic, spectroscopic and redox properties of the H-cluster. In particular, mutation of the non-conserved serine 267, situated between the [4Fe-4S] and [2Fe] subclusters, to methionine (conserved in prototypical catalytic enzymes) gave a dramatic decrease in activity. Infra-red (IR) spectroelectrochemistry revealed a 50 mV lower redox potential for the [4Fe-4S] subcluster in the S267M variant. We speculate that this serine forms a hydrogen bond to the [4Fe-4S] subcluster, increasing its redox potential. These results demonstrate the importance of the secondary coordination sphere in tuning the catalytic properties of the H-cluster in [FeFe] hydrogenases and reveal a particularly important role for amino acids interacting with the [4Fe-4S] subcluster. Site-directed mutagenesis of the sensory [FeFe] hydrogenase from Thermotoga maritima reveals new insight into how the protein environment tunes the active site properties for its sensory role.