Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor?

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.