Extrinsic 33-kilodalton protein of spinach oxygen-evolving complexes: kinetic studies of folding and disulfide reduction.
The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.
Tanaka
S
S
Department of Biology, Faculty of Science, Osaka University, Japan.
Kawata
Y
Y
Wada
K
K
Hamaguchi
K
K
D055598:Chemical Phenomena; D002621:Chemistry; D002734:Chlorophyll / Q000378:metabolism; D002736:Chloroplasts / Q000032:analysis*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D008970:Molecular Weight; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000378:metabolism; D011487:Protein Conformation; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D006146:Guanidines; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D002734:Chlorophyll; D019791:Guanidine; D004229:Dithiothreitol