A Second Soluble Hox-Type NiFe Enzyme Completes the Hydrogenase Set in Thiocapsa roseopersicina BBS

Three functional NiFe hydrogenases were previously characterized in Thiocapsa roseopersicina BBS: two of them are attached to the periplasmic membrane (HynSL and HupSL), and one is localized in the cytoplasm (HoxEFUYH). The ongoing genome sequencing project revealed the presence of genes coding for another soluble Hox-type hydrogenase enzyme (hox2FUYH). Hox2 is a heterotetrameric enzyme; no indication for an additional subunit was found. Detailed comparative in vivo and in vitro activity and expression analyses of HoxEFUYH (Hox1) and the newly discovered Hox2 enzyme were performed. Functional differences between the two soluble NiFe hydrogenases were disclosed. Hox1 seems to be connected to both sulfur metabolism and dark/photofermentative processes. The bidirectional Hox2 hydrogenase was shown to be metabolically active under specific conditions: it can evolve hydrogen in the presence of glucose at low sodium thiosulfate concentration. However, under nitrogen-fixing conditions, it can oxidize H₂ but less than the other hydrogenases in the cell.Hydrogenases are metalloenzymes involved in microbial hydrogen metabolism. A great variety of them have been identified and studied in various microorganisms and grouped on the basis of their metal content as NiFe, FeFe, and iron-sulfur cluster free hydrogenases (10, 42, 43). The basic protein structure of NiFe hydrogenases is heterodimeric, while FeFe hydrogenases are mostly composed of a single amino acid chain with multiple iron-sulfur clusters (28, 43, 44). Well-defined maturation proteins assist for the assembly and activation of hydrogenase enzymes; NiFe hydrogenases require a more complex accessory machinery than FeFe enzymes (2, 3, 24).Thiocapsa roseopersicina BBS is a photosynthetic purple sulfur bacterium belonging to the Chromatiaceae family (4). It prefers to utilize reduced sulfur compounds for anaerobic photochemolithoautotrophic growth, but simple organic substrates such as glucose or acetate can be also used as extra carbon, energy, and electron sources. It can be cultivated under aerobic (nonphotosynthetic) conditions in the presence of organic compounds. In the absence of other nitrogen sources, it is able to fix molecular nitrogen; this process is accompanied by H₂ production. T. roseopersicina was earlier shown to possess at least three NiFe hydrogenases varying in their in vivo functions, localizations, and compositions. Hyn and Hup hydrogenases are attached to the membrane facing the periplasmic side (6, 18, 30). Hyn is a bidirectional enzyme with extraordinary stability (17). Recent study has demonstrated that the HynSL subunits are physiologically connected to cellular redox processes via the Isp1 and Isp2 proteins, which play an essential role in electron transfer (27). The second membrane-associated enzyme, Hup, is involved in H₂ oxidation and shows homology to uptake hydrogenases, which recycle H₂ produced by the nitrogenase enzyme complex or present in the environment. Next to the hydrogenase small and large subunits (HupSL), a b-type cytochrome, HupC, was demonstrated to be part of the in vivo active enzyme as a transmitter of electrons to the quinone pool (27). In several bacteria, e.g., Rhodobacter capsulatus (7) and Ralstonia eutropha (15, 20), the expression of the hydrogenase(s) was shown to be regulated by the hydrogen level in the environment. The genes encoding the hydrogen-sensing system also exist in T. roseopersicina (hupUV, hupT, and hupR), but the hupTUV genes proved to be silent in the wild-type strain—only hupR is expressed—which is why expression of hupSL genes is constitutive (16).A Hox-type soluble hydrogenase was also identified in T. roseopersicina (31); it is a representative of the bidirectional heteromultimeric cytoplasmic NiFe hydrogenases (37, 39). Enzymes belonging to this group are basically composed of two moieties: hydrogenase (HoxYH) and diaphorase (HoxFU) heterodimers. Additional subunits were identified in few cases. In R. eutropha H16, two HoxI proteins completing the Hox complex were suggested to provide a binding domain for NADPH (5). HoxE has been identified as the fifth subunit of heteropentameric NAD⁺-reducing Hox hydrogenases in several cyanobacteria, Allochromatium vinosum and T. roseopersicina (21, 31, 37). In-frame deletion of the hoxE gene ceased both the H₂-producing and -oxidizing activities of Hox in vivo, but these were not affected in vitro. Consequently, an electron transfer role of the HoxE subunit was suggested (31, 32).The possibility of the presence of further hydrogenases in T. roseopersicina was noted few years ago (31). In the hynSL hupSL hoxH triple-mutant strain ({"type":"entrez-nucleotide","attrs":{"text":"GB112131","term_id":"336599610","term_text":"GB112131"}}GB112131), a small in vivo and in vitro hydrogenase activity could be measured under photomixotrophic growth conditions (both CO₂ and organic compounds are used for growth) at the late growth phase. This residual activity could not be detected in the hypF mutant strain (M539). Since HypF protein has an essential role in the maturation process of all NiFe hydrogenases (9), these results suggested the presence of a previously unknown hydrogenase. Here we describe the identification and characterization of the second Hox-type hydrogenase, emphasizing the functional similarities and differences between the two soluble enzymes of this bacterium. In order to distinguish between the two Hox-type enzymes unequivocally, the HoxEFUYH complex will be renamed Hox1 and the newly described Hox2FUYH enzyme is called Hox2.