A cyanobacterial type, heteropentameric NAD+ reducing [NiFe] hydrogenase in the purple sulfur photosynthetic bacterium, Thiocapsa roseopersicina.

Gábor Rákhely, Ákos T. Kovács, Gergely Maróti, Barna Fodor, Gyula Csanádi, Dóra Latinovics, Kornél L. Kovács

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Abstract

Structural genes coding for two membrane-associated NiFe hydrogenases in the phototrophic purple sulfur bacterium Thiocapsa roseopersicina (hupSL and hynSL) have recently been isolated and characterized. Deletion of both hydrogenase structural genes did not eliminate hydrogenase activity in the cells, and considerable hydrogenase activity was detected in the soluble fraction. The enzyme responsible for this activity was partially purified, and the gene cluster coding for a cytoplasmic, NAD+-reducing NiFe hydrogenase was identified and sequenced. The deduced gene products exhibited the highest similarity to the corresponding subunits of the cyanobacterial bidirectional soluble hydrogenases (HoxEFUYH). The five genes were localized on a single transcript according to reverse transcription-PCR experiments. A σ54-type promoter preceded the gene cluster, suggesting that there was inducible expression of the operon. The Hox hydrogenase was proven to function as a truly bidirectional hydrogenase; it produced H2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N2. In-frame deletion of the hoxE gene eliminated hydrogen evolution derived from the Hox enzyme in vivo, although it had no effect on the hydrogenase activity in vitro. This suggests that HoxE has a hydrogenase-related role; it likely participates in the electron transfer processes. This is the first example of the presence of a cyanobacterial-type, NAD+-reducing hydrogenase in a phototrophic bacterium that is not a cyanobacterium. The potential physiological implications are discussed.

Hydrogenases catalyze the simple redox reaction H2 ↔ 2H+ + 2e−. These enzymes are grouped on the basis of their metal content; some hydrogenases contain only iron atoms (Fe hydrogenases), but the majority of known hydrogen-activating enzymes contain nickel and iron at the active center (some NiFe hydrogenases also contain selenium), and one enzyme that displays hydrogenase activity in methanogens has been shown to contain no redox-active metal at all (29, 38, 40). The protein core of the NiFe hydrogenases is composed of at least two subunits. The small subunit harbors the electron-transferring Fe-S clusters, and the large subunit contains the heterobinuclear NiFe metallocenter. In the active center the Fe is coordinated by one CO and two CN ligands (39). Biosynthesis of an active hydrogenase involves complex posttranslational processing, which includes assembly of the NiFe centers, insertion of the CO and CN ligands, proteolytic cleavage of the C-terminal end of the large subunit by an endoprotease, and biosynthesis and orientation of the Fe-S clusters (9, 23). This maturation process requires the concerted action of several accessory proteins.

Hydrogenases may differ in electron carrier specificity, in cellular localization, and in regulation of expression. Hydrogenases are involved in energy conservation, in the disposal of excess electrons formed during fermentation processes, or in hydrogen sensing as a component of an H2-dependent molecular signal transduction cascade, which regulates the expression of many hydrogenases (14, 29, 38.)

In a number of species, more than one hydrogenase has been described. For example, in Escherichia coli there are four membrane-associated hydrogenases (two belonging to one group and two belonging to another group) (3, 29, 38). In Ralstonia eutropha one membrane-bound hydrogenase, one heterotetrameric cytoplasmic NAD+-reducing hydrogenase, and one regulatory hydrogenase have been described, and each of these enzymes belongs to a distinct NiFe hydrogenase family (14, 29, 38). Cyanobacteria have a special type of NAD+-reducing hydrogenases, in which an additional subunit, HoxE, is a member of the enzyme complex (HoxEFUYH) (34, 36). Most microorganisms apparently contain designated hydrogenases for diverse physiological tasks, but cross-reactions may occur as well (29).

Thiocapsa roseopersicina BBS is a purple sulfur photosynthetic bacterium (4). Two membrane-bound hydrogenases (HupSL and HynSL) have been identified in this microorganism (10, 28); both of these enzymes belong to the same group (29, 38). HynSL, which was characterized at the protein and gene levels in detail previously (19, 20, 28), is an unusually stable enzyme. It is remarkably active at high temperatures and is resistant to oxygen, proteases, and detergents (19). The organization of the hynSL genes is also extraordinary, as the genes of the small and large subunits are separated by two open reading frames (isp1 and isp2), which seem to code for the components of a transmembrane redox complex (28). No hydrogenase accessory gene was found in the vicinity of hynSL, while downstream of hupSL several genes homologous to specific accessory genes were identified (10). Seven genes coding for accessory proteins involved in the biosynthesis of hydrogenases were isolated after transposon mutagenesis in the genome of T. roseopersicina (13, 24). These genes are clustered in various loci.

The physiological role of the HynSL hydrogenase is not known, but this enzyme is believed to play a role in the maintenance of the redox balance of the cells. The other hydrogenase (HupSL) has been characterized mainly at the gene level (10), since it is difficult to purify. The putative hupSL gene products resemble the uptake hydrogenases of other microbes, which recycle the hydrogen produced by the nitrogenase during nitrogen fixation.

To establish the physiological functions of the HynSL and HupSL hydrogenases, their structural genes were deleted in the present study. Surprisingly, the cells still had hydrogenase activity, which was localized in the cytoplasm. Characterization of a gene cluster which encodes a heteropentameric NAD+-reducing hydrogenase related to the cyanobacterial bidirectional hydrogenases is described below.

(Preliminary results were presented at the Biohydrogen 2002 Conference, Ede-Wageningen, The Netherlands, 21 to 24 April 2002, and have been reviewed by Kovács et al. [20a].)
Original languageEnglish
JournalApplied and Environmental Microbiology
Volume70
Issue number2
Pages (from-to)722-728
ISSN0099-2240
DOIs
Publication statusPublished - 2004
Externally publishedYes

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