Abstract
Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.
Highlights
Ferredoxins are small proteins containing non-heme iron as iron-sulfur (FeS) clusters and they serve as electron carriers within the cell (Bruschi and Guerlesquin, 1988; Beinert, 1990)
A phylogenetic tree shows that YsaA and HydN are most closely related among all known ferredoxin-like proteins in E. coli (Figure 1)
It is conceivable that HydN and YsaA could contribute to the cofactor activation of the Formate dehydrogenase H (FDH-H) protein, since nitrate reductase (NAR) and formate dehydrogenase (FDH-N) activities are not impaired in the mutant; it would be expected that a more pronounced phenotype of the individual mutants would be observed, making this explanation unlikely
Summary
Ferredoxins are small proteins containing non-heme iron as iron-sulfur (FeS) clusters and they serve as electron carriers within the cell (Bruschi and Guerlesquin, 1988; Beinert, 1990). In addition to the ferredoxin protein itself, Fdx, which is involved in FeS cluster synthesis, ferredoxin-like proteins often have functions as small or β-subunits of modular respiratory complexes like the E. coli proteins NarH of the nitrate reductase, HybA of the H2-oxidizing hydrogenase 2, FdoH and FdnH of the formate dehydrogenases O and N, respectively, NrfC of the periplasmic nitrite reductase, HycB and HycF of the H2-producing formate hydrogenlyase (FHL) complex and HyfA of the FHL-homologous complex, FHL-2. These proteins are predicted to have a similar core-fold with alpha-antiparallel beta sandwiches to hold the FeS-clusters (Pfam domain Fer). Other ferredoxin-like proteins like AegA and YsaA are not located within or close to an operon encoding an enzyme with predicted oxidoreductase activity and no phenotype has yet been described for mutants lacking these genes (Cavicchioli et al, 1996)
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