The Physiological Functions and Structural Determinants of Catalytic Bias in the [FeFe]-Hydrogenases CpI and CpII of Clostridium pasteurianum Strain W5

Abstract

The first generation of biochemical studies of complex, iron-sulfur-cluster-containing [FeFe]-hydrogenases and Mo-nitrogenase were carried out on enzymes purified from Clostridium pasteurianum (strain W5). Previous studies suggested that two distinct [FeFe]-hydrogenases are expressed differentially under nitrogen-fixing and non-nitrogen-fixing conditions. As a result, the first characterized [FeFe]-hydrogenase (CpI) is presumed to have a primary role in central metabolism, recycling reduced electron carriers that accumulate during fermentation via proton reduction. A role for capturing reducing equivalents released as hydrogen during nitrogen fixation has been proposed for the second hydrogenase, CpII. Biochemical characterization of CpI and CpII indicated CpI has extremely high hydrogen production activity in comparison to CpII, while CpII has elevated hydrogen oxidation activity in comparison to CpI when assayed under the same conditions. This suggests that these enzymes have evolved a catalytic bias to support their respective physiological functions. Using the published genome of C. pasteurianum (strain W5) hydrogenase sequences were identified, including the already known [NiFe]-hydrogenase, CpI, and CpII sequences, and a third hydrogenase, CpIII was identified in the genome as well. Quantitative real-time PCR experiments were performed in order to analyze transcript abundance of the hydrogenases under diazotrophic and non-diazotrophic growth conditions. There is a markedly reduced level of CpI gene expression together with concomitant increases in CpII gene expression under nitrogen-fixing conditions. Structure-based analyses of the CpI and CpII sequences reveal variations in their catalytic sites that may contribute to their alternative physiological roles. This work demonstrates that the physiological roles of CpI and CpII are to evolve and to consume hydrogen, respectively, in concurrence with their catalytic activities in vitro, with CpII capturing excess reducing equivalents under nitrogen fixation conditions. Comparison of the primary sequences of CpI and CpII and their homologs provides an initial basis for identifying key structural determinants that modulate hydrogen production and hydrogen oxidation activities.

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“…Rank abundance plot for all genome bins reconstructed from the SJ3 metagenome. Genome bins are colour coded to reflect the presence of genes coding for catalytic subunits of [NiFe]‐hydrogenase or [FeFe]‐hydrogenase (HydA) that are predicted to be physiologically or catalytically biased respectively, towards the oxidation of hydrogen (H 2 ), the production of H 2 , or that encode isoforms of both enzyme types, as predicted based on phylogenetic affiliations (for [NiFe]‐hydrogenases) or presence of key catalytic residues thought to confer catalytic bias (for HydA) as reported previously (Greening et al ., ; Therien et al ., ). Many bins have multiple hydrogenases as detailed in Supporting Information Data set S2.…”

“…homology alone. The same is true for [FeFe]-hydrogenases, and only in rare cases can enzyme directionality be inferred based on homology at the primary sequence level [described below, (Therien et al, 2017)]. Nonetheless, inclusion of group 4 [NiFe]-hydrogenase and [FeFe]-hydrogenase homologues in this calculation does not change the overall pattern of enrichment of hydrogenase protein homologues in this spring type ( Fig.…”

“…The hydrogenases identified in the SJ3 bins were further classified based on homology to biochemically characterized hydrogenases and using previous classification schemes (Vignais et al, 2001;Greening et al, 2015;Poudel et al, 2016;Therien et al, 2017). The large subunits of the [NiFe]-hydrogenase were designated as belonging to subgroups of group 1, 2, 3 or 4 [NiFe]-hydrogenases, based on phylogenetic relationships (Greening et al, 2015) ( Fig.…”

“…6B). [NiFe]-hydrogenase homologues affiliated with groups 1, 2 and 3 are physiologically biased towards H 2 oxidation or are bidirectional (Vignais et al, 2001). For [FeFe]hydrogenases, catalytic bias was predicted based on conservation of residues in the active site H-cluster domain of the catalytic subunit, HydA, when compared to homologues from C. pasteurianum which were previously shown to be catalytically biased towards H 2 oxidation (CpII) or production (CpI) (Adams and Mortenson, 1984;Therien et al, 2017). The primary ligands for the H cluster and accessory [iron (Fe)-sulphur (S)] clusters in HydA are conserved (Meyer, 2007), indicating that these are not responsible for catalytic bias.…”

“…The primary ligands for the H cluster and accessory [iron (Fe)-sulphur (S)] clusters in HydA are conserved (Meyer, 2007), indicating that these are not responsible for catalytic bias. However, key residues in the secondary coordination sphere of the H cluster and accessory [Fe-S] clusters of HydA can be variable and have been suggested to contribute to catalytic bias (Therien et al, 2017). For example, 13 of the 14 HydA in SJ3 bins (sole exception being HydA in Bin 98) exhibit substitutions in key secondary coordination sphere residues, including position 163 (Fe sub-cluster of the active site H cluster), position 268 (proximal 4Fe-4S cluster), and positions 357/505 (4Fe-4S-subcluster of the H cluster), that have been suggested to catalytically bias HydA towards H 2 oxidation [Supporting Information Fig.…”