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 --> His/Phe-43 --> His, called Cu(B)Mb) that closely mimics the heme-copper center in HCO. In the absence of metal ions, the engineered CUB center in CU(B)Mb decreases the O-2 binding affinity of the heme. However, addition of Ag(1), a redox-inactive mimic of Cu(I), increases the O-2-binding affinity. More importantly, copper ion in the CUB center is essential for O-2 reduction, as no O-2 reduction can be observed in copper-free, Zn(II), or Ag(1) derivatives of CUBMb. Instead of producing a ferryl-heme as in HCO, the CuBMb generates verdoheme because the engineered CuBMb may lack a hydrogen bonding network that delivers protons to promote the heterolytic O-O cleavage necessary for the formation of ferryl-heme. Reaction of oxidized CuBMb with H2O2, 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 CUB 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.