A site-directed mutagenesis study on the conserved subunit I histidines of the cytochrome bo complex in Escherichia coli identified ligands of the low spin heme B and CuB centers; however, the assignment of the proximal ligand of the high spin heme O was ambiguous (Minagawa, J., Mogi, T., Gennis, R. B., and Anraku, Y. (1992) J. Biol. Chem. 267, 2096-2104). We have extended this work and characterized the metal centers in the purified histidine mutant oxidases by optical, EPR, and resonance Raman spectroscopies and by biochemical analysis. We found that the H284A and H333A oxidases contain two heme B molecules, which exhibit the gz = 2.99 low spin and cyanide-sensitive g perpendicular = 6 high-spin EPR signals, whereas the H419A oxidase contains only low spin heme B, which shows the gz component in a considerably higher magnetic field at g = 2.92. The CuB center was partially retained in the H284A oxidase but was almost completely lost in the H333A and H419A oxidases. Thus, we concluded that His419 is the proximal ligand of the high spin heme O and that His284 is located at the distal side of the high spin heme O. His284 plays an indispensable role in maintaining the structure of the Feo-CuB binuclear site suitable for the exogenous ligand bindings in the reduced state, since its substitution eliminated the CO binding activity. In addition, we found that His106 and His421 are in fact the axial ligands of the low spin heme B, although the H421A mutation perturbed the binuclear metal center seriously. Based upon experimental results with isotopic substitutions on iron in the oxidases, we assigned the Raman band at 208 cm-1 to the iron-histidine stretching mode (vFc(2+)-N(His)) of the wild-type ferrous cytochrome o, which was upshifted slightly by a loss of the CuB center in the H333A oxidase. A molecular structure of the metal centers and a possible mechanism of the electron transfer-coupled proton pumping in the cytochrome bo complex are proposed on the basis of our present findings.
Copper Cytochrome b Group