PURIFICATION OF RHIZOBIUM-LEGUMINOSARUM HYPB, A NICKEL-BINDING PROTEIN REQUIRED FOR HYDROGENASE SYNTHESIS

The products of the Rhizobium leguminosarum hyp gene cluster are neces sary for synthesis of a functional uptake [NiFe] hydrogenase system in symbiosis with pea plants, and at least for HgpB and HypF, a role in hydrogenase-specific nickel metabolism has been postulated (L. Rey, J. Murillo, Y. Hernando, E. Hidalgo, E. Cabrera, J. Imperial, and T. Rui z-Argueso, Mol. Microbiol. 8:471-481, 1993). The R. leguminosarum hypB gene product has been overexpressed in Escherichia coil and purified by immobilized nickel chelate affinity chromatography in a single step . The purified recombinant HypB protein was able to bind 3.9 +/- 0.1 N i2+ ions per HgpB monomer in solution. Co2+, CU2+, and Zn2+ ions compe ted with Ni2+ with increasing efficiency. Monospecific HypB antibodies were raised and used to show that HypB is synthesized in R. leguminos arum microaerobic vegetative cells and pea bacteroids but not in R. le guminosarum aerobic cells. HypB protein synthesized by R. leguminosaru m microaerobic vegetative cells could also be isolated by immobilized nickel chelate affinity chromatography. A histidine-rich region at the amino terminus of the protein (23-HGHHHH DGHHDHDHDHDHHRGDHEHDDHHH-54) is proposed to play a role in nickel binding, both in solution and in chelated form.