Copper X-ray absorption spectroscopy (XAS) has been used to examine the structures of the Cu(II) and Cu(I) forms of the cytochrome bo3 quinol oxidase from Escherichia coli. Cytochrome bo3 is a member of the superfamily of heme-copper respiratory oxidases. Of particular interest is the fact that these enzymes function as redox-linked proton pumps, resulting in the net translocation of one H+ per electron across the membrane. The molecular mechanism of how this pump operates and the manner by which it is linked to the oxygen chemistry at the active site of the enzyme are unknown. Several proposals have featured changes in the coordination of Cu(B) during enzyme turnover that would result in sequential protonation or deprotonation events that are key to the functioning proton pump. This would imply lability of the ligands to Cu(B). In this work, the structure of the protein in the immediate vicinity of Cu(B), in both the fully oxidized and fully reduced forms of the enzyme, has been examined by Cu XAS, a technique that is particularly sensitive to changes in metal coordination. The results show that in the oxidized enzyme, Cu(B)(II) is four-coordinate, consistent with three imidazoles and one hydroxyl (or water). Upon reduction of the enzyme, the coordination of Cu(B)(I) is significantly altered, consistent with the loss of one of the histidine imidazole ligands in at least a substantial fraction of the population. These data add to the credibility that changes in the ligation of Cu(B) might occur during catalytic turnover of the enzyme and, therefore, could, in principle, be part of the mechanism of proton pumping.
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