The role of copper and protons in heme-copper oxidases: kinetic study of an engineered heme-copper center in myoglobin.

To probe the role of copper and protons in heme-copper oxidase (HCO), we have performed kinetic studies on an engineered heme-copper center in sperm whale myoglobin (Leu-29 --> HisPhe-43 --> His, called Cu(B)Mb) that closely mimics the heme-copper center in HCO. In the absence of metal ions, the engineered Cu(B) center in Cu(B)Mb decreases the O(2) binding affinity of the heme. However, addition of Ag(I), a redox-inactive mimic of Cu(I), increases the O(2)-binding affinity. More importantly, copper ion in the Cu(B) center is essential for O(2) reduction, as no O(2) reduction can be observed in copper-free, Zn(II), or Ag(I) derivatives of Cu(B)Mb. Instead of producing a ferryl-heme as in HCO, the Cu(B)Mb generates verdoheme because the engineered Cu(B)Mb may lack a hydrogen bonding network that delivers protons to promote the heterolytic OO cleavage necessary for the formation of ferryl-heme. Reaction of oxidized Cu(B)Mb with H(2)O(2), a species equivalent in oxidation state to 2e(-), reduced O(2) but, possessing the extra protons, resulted in ferryl-heme formation, as in HCO. The results showed that the Cu(B) center plays a critical role in O(2) binding and reduction, and that proton delivery during the O(2) reduction is important to avoid heme degradation and to promote the HCO reaction.