Abstract
The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H2 or other reduced products has been limited by their sensitivity to oxygen (O2). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O2 gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O2 concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H2 transfer and O2 susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O2 tolerant enzymes for biotechnological applications.
Highlights
Hydrogen (H2) is a diffusible electron carrier with the highest energy content per unit mass of all naturally occurring fuels, and forms the basis of numerous interspecies interactions in natural microbial communities [1,2]. [NiFe]- and [FeFe]-hydrogenase, which differ in the metal composition of their respective active-site clusters, are principally responsible for H2 cycling in natural environments
A diversity of [FeFe]-hydrogenase were identified along the DWR3 vertical gradient in the Great Salt Lake (GSL)
GSL form trimers and are possibly involved in H2-based electron bifurcation; it is unclear if they are involved in the production or consumption of H2
Summary
Hydrogen (H2) is a diffusible electron carrier with the highest energy content per unit mass of all naturally occurring fuels, and forms the basis of numerous interspecies interactions in natural microbial communities [1,2]. [NiFe]- and [FeFe]-hydrogenase, which differ in the metal composition of their respective active-site clusters, are principally responsible for H2 cycling in natural environments. Characterization of [FeFe]-hydrogenase homologs in available genome sequences [3,19] noted substantial variation in N- and C-terminal Fe-S cluster and accessory cofactor binding motifs, suggesting potential interactions with a variety of other redox partners Such analyses performed in conjunction with an evaluation of gene context and biochemical characterization revealed differences in [FeFe]-hydrogenase quaternary structure, including the identification of multimeric homologs that likely form dimers, trimers, or tetramers [3,19]. A previous application of a PCR-directed approach targeting a fragment of the gene encoding the large subunit (hydA) of [FeFe]-hydrogenase in a phototrophic mat community identified significant variation in the amino acid environment of the active site H-cluster [41] and previously implicated gas channels [39,45,46,47,48]. This study demonstrates that enzyme variants with desired properties can potentially be recovered by examining protein diversity in microbial assemblages that have evolved in the presence of a particular environmental stress
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