Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis: An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase

Princess R Cabotaje; Kaija Walter; Afridi Zamader; Ping Huang; Felix Ho; Henrik Land; Moritz Senger; Gustav Berggren

doi:10.1021/acscatal.3c02314

Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis: An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase

ACS Catal. 2023 Jul 26;13(15):10435-10446. doi: 10.1021/acscatal.3c02314. eCollection 2023 Aug 4.

Authors

Princess R Cabotaje¹, Kaija Walter¹, Afridi Zamader¹, Ping Huang¹, Felix Ho¹, Henrik Land¹, Moritz Senger¹, Gustav Berggren¹

Affiliation

¹ Molecular Biomimetics, Department of Chemistry, Ångström Laboratory, Uppsala University, Box 523, SE-75120 Uppsala, Sweden.

Abstract

[FeFe] hydrogenases, metalloenzymes catalyzing proton/dihydrogen interconversion, have attracted intense attention due to their remarkable catalytic properties and (bio-)technological potential for a future hydrogen economy. In order to unravel the factors enabling their efficient catalysis, both their unique organometallic cofactors and protein structural features, i.e., "outer-coordination sphere" effects have been intensively studied. These structurally diverse enzymes are divided into distinct phylogenetic groups, denoted as Group A-D. Prototypical Group A hydrogenases display high turnover rates (10⁴-10⁵ s^-1). Conversely, the sole characterized Group D representative, Thermoanaerobacter mathranii HydS (TamHydS), shows relatively low catalytic activity (specific activity 10^-1 μmol H₂ mg^-1 min^-1) and has been proposed to serve a H₂-sensory function. The various groups of [FeFe] hydrogenase share the same catalytic cofactor, the H-cluster, and the structural factors causing the diverging reactivities of Group A and D remain to be elucidated. In the case of the highly active Group A enzymes, a well-defined proton transfer pathway (PTP) has been identified, which shuttles H⁺ between the enzyme surface and the active site. In Group D hydrogenases, this conserved pathway is absent. Here, we report on the identification of highly conserved amino acid residues in Group D hydrogenases that constitute a possible alternative PTP. We varied two proposed key amino acid residues of this pathway (E252 and E289, TamHydS numbering) via site-directed mutagenesis and analyzed the resulting variants via biochemical and spectroscopic methods. All variants displayed significantly decreased H₂-evolution and -oxidation activities. Additionally, the variants showed two redox states that were not characterized previously. These findings provide initial evidence that these amino acid residues are central to the putative PTP of Group D [FeFe] hydrogenase. Since the identified residues are highly conserved in Group D exclusively, our results support the notion that the PTP is not universal for different phylogenetic groups in [FeFe] hydrogenases.