pH-induced conformational transition in the soluble CuA domain of Paracoccus denitrificans cytochrome oxidase

Biochemistry. 2001 May 22;40(20):6180-9. doi: 10.1021/bi002212e.

Abstract

The pH-induced conformational transition in the CuA domain of subunit II of cytochrome oxidase of Paracoccus denitrificans (PdII) has been investigated using various spectroscopic and stopped-flow kinetic methods. UV-visible absorption and circular dichroism studies showed that an increase in pH from 6 to 10 leads to a conformation change with pK(a) = 8.2 associated with the CuA site of the protein. The secondary structure of the protein was, however, shown to remain unchanged in these two conformational states. Thermal and urea-induced unfolding studies showed that the "low-pH" conformation is more stable compared to the "high-pH" conformation of the protein. Moreover, the overall stability of the protein was found to decrease on reduction of the metal centers in the low-pH form, while the oxidation state of the metal centers did not have any significant effect on the overall stability of the protein in the high-pH form. Stopped-flow pH-jump kinetic studies suggested that the conformational transition is associated with a slow deprotonation step followed by fast conformational equilibrium. The results are discussed in the light of understanding the pH-induced conformational change in the beta-barrel structure of the protein and its effect on the coordination geometry of the metal site.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Circular Dichroism
  • Copper / chemistry*
  • Electron Transport Complex IV / chemistry*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Oxidation-Reduction
  • Paracoccus denitrificans / enzymology*
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Protein Structure, Tertiary
  • Solubility
  • Spectrophotometry, Ultraviolet
  • Temperature
  • Thermodynamics
  • Urea / chemistry

Substances

  • Copper
  • Urea
  • Electron Transport Complex IV