Electrochemical determination of heme-linked pKa values and the importance of using fluoride binding in heme proteins

Anal Biochem. 2013 Dec 1;443(1):75-7. doi: 10.1016/j.ab.2013.08.016. Epub 2013 Aug 23.

Abstract

The ultraviolet-visible (UV-vis) spectroelectrochemical measurements of heme proteins in the presence of a heme-bound fluoride ion can be used as a probe for heme-linked ionizations of acid-base groups in the heme pocket. A detailed study of the pH dependence of the midpoint potential of skeletal horse myoglobin (Mb) with a heme-bound fluoride ion (Mb-F) reveals how protonation of the distal histidine (H64) changes the redox properties of the protein with a determined pKa of 5.3. In addition, fluoride binding in myoglobin provides a stabilization of -1.9 kcal/mol of the ferric Mb-F relative to ferric Mb without fluoride.

Keywords: Fluoride; Midpoint potential; Myoglobin; Spectroelectrochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Electrochemical Techniques
  • Electron Transport
  • Fluorides / chemistry*
  • Heme / analysis
  • Heme / chemistry*
  • Histidine / chemistry*
  • Horses
  • Hydrogen-Ion Concentration
  • Kinetics
  • Muscle, Skeletal / chemistry
  • Myoglobin / analysis
  • Myoglobin / chemistry*
  • Oxidation-Reduction
  • Protein Binding
  • Protons*
  • Spectrophotometry / methods
  • Thermodynamics

Substances

  • Myoglobin
  • Protons
  • Heme
  • Histidine
  • Fluorides