JBIC Journal of Biological Inorganic Chemistry

, Volume 18, Issue 6, pp 595–598 | Cite as

Building reactive copper centers in human carbonic anhydrase II

  • He Song
  • Andrew C. Weitz
  • Michael P. Hendrich
  • Edwin A. Lewis
  • Joseph P. Emerson
Report

Abstract

Reengineering metalloproteins to generate new biologically relevant metal centers is an effective a way to test our understanding of the structural and mechanistic features that steer chemical transformations in biological systems. Here, we report thermodynamic data characterizing the formation of two type-2 copper sites in carbonic anhydrase and experimental evidence showing one of these new, copper centers has characteristics similar to a variety of well-characterized copper centers in synthetic models and enzymatic systems. Human carbonic anhydrase II is known to bind two Cu2+ ions; these binding events were explored using modern isothermal titration calorimetry techniques that have become a proven method to accurately measure metal-binding thermodynamic parameters. The two Cu2+-binding events have different affinities (Ka approximately 5 × 1012 and 1 × 1010), and both are enthalpically driven processes. Reconstituting these Cu2+ sites under a range of conditions has allowed us to assign the Cu2+-binding event to the three-histidine, native, metal-binding site. Our initial efforts to characterize these Cu2+ sites have yielded data that show distinctive (and noncoupled) EPR signals associated with each copper-binding site and that this reconstituted enzyme can activate hydrogen peroxide to catalyze the oxidation of 2-aminophenol.

Supplementary material

775_2013_1009_MOESM1_ESM.pdf (241 kb)
Experimental details as well as Figs. A, B, and C can be found in the electronic supplementary material. Supplementary material 1 (PDF 240 kb)

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Copyright information

© SBIC 2013

Authors and Affiliations

  • He Song
    • 1
  • Andrew C. Weitz
    • 2
  • Michael P. Hendrich
    • 2
  • Edwin A. Lewis
    • 1
  • Joseph P. Emerson
    • 1
  1. 1.Department of Chemistry, 1115 Hand LabMississippi State UniversityMississippi StateUSA
  2. 2.Department of ChemistryCarnegie Mellon UniversityPittsburghUSA

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