SPECTROSCOPIC CHARACTERIZATION OF MUTANTS SUPPORTS THE ASSIGNMENT OF HISTIDINE-419 AS THE AXIAL LIGAND OF HEME-O IN THE BINUCLEAR CENTER OFTHE CYTOCHROME-BO UBIQUINOL OXIDASE FROM ESCHERICHIA-COLI

The bo-type ubiquinol oxidase of Escherichia coli is a member of the s uperfamily of heme-copper oxidases which also includes the aa3-type cy tochrome c oxidases. The oxygen-binding binuclear center of cytochrome bo is located in subunit I and consists of a heme (heme o; heme a3 in the aa3-type oxidases) and a copper (Cu(B)). Previous spectroscopic s tudies have shown that heme o is bound to the protein via a single his tidine residue. Site-directed mutagenesis of conserved histidine resid ues in subunit I has identified two residues (H284 and H419) which are candidates for the ligand of heme o, while spectroscopic studies of m utants at H284 definitively demonstrated that this residue cannot be t he axial ligand. Consequently, the single remaining conserved histidin e in subunit I (H419) was assigned as the ligand for the heme of the b inuclear center. In this paper, this assignment is tested by character ization of additional mutants in which the putative heme o axial ligan d, H419, is replaced by other amino acids. All mutations at H419 resul t in the loss of enzyme activity. Analyses via UV-visible and Fourier transform infrared spectroscopies reveal that substantial perturbation has occurred at the binuclear center as a result of the amino acid su bstitutions. In contrast with the wild-type enzyme, the mutant enzymes bind very little carbon monoxide. Three other amino acid residues whi ch are potential ligands for heme o are shown to be nonessential for e nzyme activity. Mutations in these residues do not perturb the UV-visi ble or FTIR spectroscopic characteristics of the enzyme. These results are consistent with the assignment of H419 as the axial ligand of hem e o in the E. coli enzyme and, by analogy, heme a3 in the aa3-type cyt ochrome c oxidases.