Cu XAS shows a change in the ligation of Cu-B upon reduction of cytochromebo(3) from Escherichia coli

Copper X-ray absorption spectroscopy (XAS) has been used to examine the str uctures of the Cu(II) and Cu(I) forms of the cytochrome bos quinol oxidase from Escherichia coli. Cytochrome bos is a member of the superfamily of hem e-copper respiratory oxidases. Of particular interest is the fact that thes e enzymes function as redox-linked proton pumps, resulting in the net trans location of one H+ per electron across the membrane. The molecular mechanis m of how this pump operates and the manner by which it is linked to the oxy gen chemistry at the active site of the enzyme are unknown. Several proposa ls have featured changes in the coordination of Cu-B during enzyme turnover that would result in sequential protonation or deprotonation events that a re key to the functioning proton pump. This would imply lability of the lig ands to Cu-B. In this work, the structure of the protein in the immediate v icinity 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 sensi tive to changes in metal coordination. The results show that in the oxidize d 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 h istidine imidazole ligands in at least a substantial fraction of the popula tion. 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.