Identification of metal-binding residues in the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is p roposed to bind intracellular Ni(II) for transfer to urease apoprotein, Whi le native UreE possesses a histidine-rich region at its carboxyl terminus t hat binds several equivalents of Ni, the Ni-binding sites associated with u rease activation are internal to the protein as shown by studies involving truncated H144*UreE [Brayman and Hausinger (1996) J. Bacteriol. 178, 5410-5 416]. Nine potential Ni-binding residues (five His, two Cys, one Asp, and o ne Tyr within H144*UreE were independently substituted by mutagenesis to de termine their roles in metal binding and urease activation. In vivo effects of these substitutions on urease activity were measured in Escherichia col i strains containing the K. aerogenes urease gene cluster with the mutated ureE genes. Several mutational changes led to reductions in specific activi ty, with substitution of His96 producing urease activity below the level ob tained from a ureE deletion mutant. The metal-binding properties of purifie d variant UreE proteins were characterized by a combination of equilibrium dialysis and UV/visible, EPR, and hyperfine-shifted H-1 NMR spectroscopic m ethods. Ni binding was unaffected for most H144*UreE variants, but mutant p roteins substituted at His 110 or His 112 exhibited greatly reduced affinit y for Ni and bound one, rather than two, metal ions per dimer. Cys79 was id entified as the Cu ligand responsible for the previously observed charge-tr ansfer transition at 370 nm, and His 112 also was shown to be associated wi th this chromophoric site. NMR spectroscopy provided clear evidence that Hi s96 and His110 serve as ligands to Ni or Co. The results from these and oth er studies, in combination with prior spectroscopic findings for metal-subs tituted UreE [Colpas ct al. (1998) J. Biol. Inorg. Chem. 3, 150-160], allow us to propose that the homodimeric protein possesses two nonidentical meta l-binding sites, each symmetrically located at the dimer interface. The fir st equivalent of added Ni or Co binds via His96 and His112 residues from ea ch subunit of the dimer, and two other N or O donors. Asp111 either functio ns as a ligand or may affect this site by secondary interactions. The secon d equivalent of Ni or Co binds via the symmetric pair of His110 residues as well as four other N or O donors. In contrast, the first equivalent of Cu binds via the His 110 pair and two other N/O donors, while the second equiv alent of Cu binds via the His112 pair and at least one Cys79 residue. UreE sequence comparisons among urease-containing microorganisms reveal that res idues His96 and Asp111, associated with the first site of Ni binding, are h ighly conserved, while the other targeted residues are missing in many case s. Our data are most compatible with one Ni-binding site per dimer being cr itical for UreE's function as a metallochaperone.