Purification and properties of a protein linked to the soluble hydrogenase of hydrogen-oxidizing bacteria

Abstract

In Alcaligenes eutrophus, the formation of the hydrogenases and of five new peptides is subject to the hydrogenase control system. Of these, the B peptide was purified to homogeneity. This protein (Mr, 37,500) was composed of two identical subunits (Mr,18,800). Antibodies against the B protein were used for its quantification by rocket immunoelectrophoresis. About 4% of the total protein consisted of the B protein; its molar ratio to the NAD-linked hydrogenase was about 4:1. The B protein appeared to be associated with the NAD-linked hydrogenase, as shown by gel filtration analysis with Sephadex G-200. The B protein was not detected in cells that had not expressed the hydrogenase proteins or that lacked the genetic information of the hydrogen-oxidizing character; it was also not detected in Tn5 insertional mutants that were unable to form soluble hydrogenase antigens. Immunochemical analysis of other species and genera than A. eutrophus revealed that only strains able to form a NAD-linked hydrogenase also formed B-protein antigens. The B protein is not required for the catalytic activity of soluble hydrogenase in vitro; its function is at present unknown.

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

“…We demonstrate that HoxI is identical with the so-called B protein which has been shown to be coexpressed with the SH at high levels (30). HoxI harbors a putative cyclic nucleotide-binding site in its central domain.…”

“…However, a major protein species usually coexpressed with the R. eutropha SH was missing in SDS-PAGE gels (S. Thiemermann and B. Friedrich, unpublished data). Earlier studies by Kärst et al (30) showed that several proteins are specifically synthesized under hydrogenase-derepressing conditions in R. eutropha H16. One of them, a 19-kDa species called the B protein, had been purified.…”

“…The fact that the B protein coeluted with the SH on different chromatographic media (30) prompted us to reinvestigate the purification conditions. After anion exchange chromatography on DEAE, a 19-kDa protein coeluted with the SH.…”

Genes encoding the NAD-reducing hydrogenase ofRhodococcus opacus MR11

“…This result, and the property of the B protein of H16 to form dimers in the native state (Karst et al, 1987), like the E. coli CRP and the kinases, suggests that this protein may be involved in signal transduction pathways. But interestingly, ORF2 deletion mutants of H16 revealed no recognizable phenotype (Thiemermann et al, 1996).…”

“…In strain H16, HoxW was recognized to be a protease of high specificity which removes a 24 amino acid residue peptide chain from the C-terminus of the p subunit before assembly of the four subunits occurs (Thiemermann et al, 1996). However, the function of the ORF2 gene product in H16, the so-called B protein (Karst et al, 1987), has not yet been elucidated. An alignment of the ORF7 and ORF2 gene products of R. opacus and A. eutrophus with the cGMP-and CAMPdependent kinases of cattle and Ap.…”