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

• Published in: Proceedings of the National Academy of Sciences
• Main Authors: Sigman, Jeffrey A., Kim, Hyeon K., Zhao, Xuan, Carey, James R., Lu, Yi
• Format: Text
• Language: unknown
• Published: University of Memphis Digital Commons 2003
• Subjects: Biomimetic models; Cytochrome oxidase; Protein design; Protein engineering; Chemistry; Sperm whale
• Online Access: https://digitalcommons.memphis.edu/facpubs/2270; https://doi.org/10.1073/pnas.0737308100

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 CuBMb) that closely mimics the heme-copper center in HCO. In the absence of metal ions, the engineered CuB center in CuBMb decreases the O2 binding affinity of the heme. However, addition of Ag(I), a redox-inactive mimic of Cu(I), increases the O2-binding affinity. More importantly, copper ion in the CuB center is essential for O2 reduction, as no O2 reduction can be observed in copper-free, Zn(II), or Ag(I) 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 O2 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 O2 binding and reduction, and that proton delivery during the O2 reduction is important to avoid heme degradation and to promote the HCO reaction.