Thermal denaturation of spinach plastocyanin: effect of copper site oxidation state and molecular oxygen.
The thermal denaturation of the cupredoxin plastocyanin (PC) from spinach has been studied with the aim of improving the understanding of factors involved in the conformational stability of antiparallel beta-sheet proteins. Studies using differential scanning calorimetry have been complemented with nuclear magnetic resonance spectroscopy, absorbance spectroscopy, dynamic light scattering, and mass spectrometry in elucidation of the effect of the copper-site oxidation state on the irreversible thermal denaturation process. Our results indicate that copper-catalyzed oxidation of the metal-ligating cysteine is the sole factor resulting in thermal irreversibility. However, this can be prevented in reduced protein by the removal of molecular oxygen. Application of a two-state equilibrium transition model to the folding process thus allowed the extraction of thermodynamic parameters for the reduced protein (Delta(trs)H = 494 kJ mol(-1), DeltaH(vH) = 343 kJ mol(-1), and T(m) = 71 degrees C). However, anaerobically denatured oxidized protein and all aerobically denatured species undergo covalent modification as a result of the copper-catalyzed oxidation of the metal-ligating cysteine residue resulting in the formation of both oxidized monomers and disulfide-linked dimers. On the basis of these results, a general mechanism for the irreversible thermal denaturation of cupredoxins is proposed. The results presented here also indicate that PC, as opposed to the previously characterized homologous protein azurin, unfolds via at least one significantly populated intermediate state (DeltaH(vH)/Delta(trs)H = 0.7) despite the almost identical native state topologies of these proteins. These findings will aid the characterization of the stability of PC and other cupredoxins and possibly of all cysteine-ligating metal-binding proteins.
Sandberg
Anders
A
Department of Chemistry, University of Göteborg, Box 462, 405 30 Göteborg, Sweden.
Harrison
David J
DJ
Karlsson
B Göran
BG
D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel / Q000379:methods; D003300:Copper / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D006358:Hot Temperature; D008027:Light; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D010970:Plastocyanin / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D018724:Spinacia oleracea / Q000737:chemistry*; D013816:Thermodynamics
D003300:Copper; D010970:Plastocyanin; D003545:Cysteine; D010100:Oxygen