Redox-dependent structural changes in an engineered heme-copper center in myoglobin: insights into chloride binding to CuB in heme copper oxidases

The effects of chloride on the redox properties of an engineered binuclear heme-copper center in myoglobin (Cu(B)Mb) were studied by UV-vis spectroelectrochemistry and EPR spectroscopy. A low-spin heme Fe(III)-Cu(I) intermediate was observed during the redox titration of Cu(B)Mb only in the presence of both Cu(II) and chloride. Upon the first electron transfer to the Cu(B) center, one of the His ligands of Cu(B) center dissociates and coordinates to the heme iron, forming a six-coordinate low-spin ferric heme center and a reduced Cu(B) center. The second electron transfer reduces the ferric heme and causes the release of the coordinated His ligand. Thus, the fully reduced state of the heme-copper center contains a five-coordinate ferrous heme and a reduced Cu(B) center, ready for O(2) binding and reduction to water to occur. In the absence of a chloride ion, formation of the low-spin heme species was not observed. These redox reactions are completely reversible. These results indicate that binding of chloride to the Cu(B) center can induce redox-dependent structural changes, and the bound chloride and hydroxide in the heme-copper center may play different roles in the redox-linked enzymatic reactions of heme-copper oxidases, probably because of their different binding affinity to the copper center and the relatively high concentration of chloride under physiological conditions.