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

Jeffrey A. Sigman, Hyeon K. Kim, Xuan Zhao, James R. Carey, Yi Lu

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)3629-3634
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number7
DOIs
StatePublished - Apr 1 2003

Keywords

  • Biomimetic models
  • Cytochrome oxidase
  • Protein design
  • Protein engineering

ASJC Scopus subject areas

  • Genetics
  • General

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