Cyanobacterial-Type, Heteropentameric, NAD ⁺ -Reducing NiFe Hydrogenase in the Purple Sulfur Photosynthetic Bacterium Thiocapsa roseopersicina

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 54 -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 H 2 under nitrogenase-repressed conditions, and it recycled the hydrogen produced by the nitrogenase in cells fixing N 2 . 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.

The Soluble NAD+-Reducing [NiFe]-Hydrogenase fromRalstonia eutrophaH16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

“…PCC 6803, Synechococcus sp. PCC 6301, and Thiocapsa roseopersicina (53,55), enzyme purification has subsequently uncovered an additional hydrogenase-related subunit designated HoxE. A HoxE homologue is predicted from the genome sequence of Nostoc sp.…”

“…Related hydrogenases are present in Rhodococcus opacus (24,56), in several species of cyanobacteria (65), and in Thiocapsa roseopersicina (53). The SH consists of four heterologous subunits forming two functional modules: a hydrogenase dimer encoded by the hoxH and hoxY genes and an NADH-dehydrogenase dimer encoded by hoxF and hoxU.…”

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

“…In organisms other than cyanobacteria, a pentameric NADH-dependent bidirectional hydrogenase is present in Thiocapsa roseopersicina (174) and in Allochromatium vinosum (108,127). The best-studied bidirectional hydrogenase, the NADH-dependent enzyme from Ralstonia eutropha, is encoded only by hoxFUYH (75).…”

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

“…HoxH and HoxY form the hydrogenase moiety and are homologous to the large and small subunits of prototypical heterodimeric [NiFe]-hydrogenases, respectively (1). The [FeS] cluster-containing subunits HoxF, HoxU, and, in some organisms, HoxE form the diaphorase moiety (13,14) that catalyzes the oxidation/reduction of NAD(P)H/ NAD(P) ϩ (via FMN and NAD binding sites in HoxF) coupled to the hydrogenase moiety (HoxYH) (10,(12)(13)(14)(15). The R. eutropha Hox hydrogenase does not contain HoxE and instead harbors an unrelated fifth subunit, HoxI, which functions in linkage to NADPH (16).…”