The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex

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.

Dissection of the Hydrogen Metabolism of the Enterobacterium Trabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I

“…Furthermore, deletion of the fdhF gene, coding for the FHL-1 Ec -associated formate dehydrogenase H, and the subsequent addition of pTg-hyf showed that the activity of the heterologous FHL-2 Tg complex was absent and thus identified FdhH as the electron input module for FHL-2 Tg in E. coli. The E. coli and T. guamensis formate dehydrogenases are both encoded at distinct locations from the operons encoding FHL, and recent work indicated that FdhH can form several protein complexes within the cell (34). The proteins share 95% amino acid identity between the two organisms, including the catalytic selenocysteine residue.…”

“…One unit of activity represents the conversion of 1 mol substrate per min. H 2 headspace content was determined with a Shimadzu-2010 gas chromatography system equipped with a ShinCarbon micropacked ST80/100 column, as described previously (34). The amount of H 2 was normalized to the OD 600 of the culture and the used culture volume.…”

Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis

“…A recent study in E. coli identified a flexible interaction network of ferredoxin-like proteins with Fdh-H, including its small, electron-transferring subunit, HycB ( Pinske, 2018 ). HycB and HupX belong to this family but share only 24% amino acid identity (38% similarity) in a MuscleWS alignment.…”

“…The ferredoxin-like family of electron–transfer proteins harbors four [4Fe-4S] clusters and an interaction network of several members of this family has been uncovered recently in E. coli ( Pinske, 2018 ). One member is HycB, the small subunit of the formate dehydrogenase (Fdh-H) that forms one of the two catalytic centers of the formate hydrogenlyase (FHL) complex, and another is the related protein HydN, which is proposed to be involved in FHL complex assembly ( Pinske, 2018 ). Generally, however, the physiological function of most members of this emerging superfamily of iron-sulfur-containing electron transfer proteins is not understood.…”

The N‐terminal domains of the paralogous HycE and NuoCD govern assembly of the respective formate hydrogenlyase and NADH dehydrogenase complexes

“…A volume of 2 mL degassed MOPS buffer, pH 7.0, was used, and the reaction was started with 15 mM formate. The H 2 content of the gas headspace of a 15-mL Hungate tube filled with 7 mL culture was determined by gas chromatography using a GC2010 Plus Gas Chromatograph (Shimadzu, Ky oto, Japan) as described [39].…”