pH-induced conformational transition in the soluble CuA domain of Paracoccus denitrificans cytochrome oxidase.
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.
Gupta
S
S
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400005, India.
Warne
A
A
Saraste
M
M
Mazumdar
S
S
D001665:Binding Sites; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D003300:Copper; D014508:Urea; D003576:Electron Transport Complex IV