Open AccessEffects of folding on metalloprotein active sites
Author(s) -
Jay R. Winkler,
Pernilla WittungStafshede,
Johan Leckner,
Bo G. Malmström,
Harry B. Gray
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.9.4246
Subject(s) - azurin , chemistry , crystallography , protein folding , tetragonal crystal system , cytochrome c , guanidinium chloride , folding (dsp implementation) , metalloprotein , aqueous solution , heme , cytochrome , biophysics , redox , inorganic chemistry , metal , crystal structure , biochemistry , organic chemistry , biology , enzyme , electrical engineering , mitochondrion , engineering
Experimental data for the unfolding of cytochromec and azurin by guanidinium chloride (GuHCl) are used to construct free-energy diagrams for the folding of the oxidized and reduced proteins. With cytochromec , the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochromec are less stable than the corresponding states of the horse protein. Due to the covalent attachment of the heme and its fixed tetragonal coordination geometry, cytochromec folding can be described by a two-state model. A thermodynamic cycle leads to an expression for the difference in self-exchange reorganization energies for the folded and unfolded proteins. The reorganization energy for electron exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein suggests that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of CuI in the unfolded protein is linear or trigonal, whereas CuII prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1, 10-phenanthroline)2 2+/+ reference system.