Abstract
The thermal stability of plastocyanin (PC) was determined as a function of oxidation state of the copper center and the presence of oxidants, reductants, oxygen, and EDTA. It was found that the copper center and its ligands play a crucial role in maintaining the stability of PC. Thermal denaturation was monitored by using far-uv circular dichroism (CD) spectra to monitor changes in secondary structure, the near-uv CD ellipticity at 280 nm to monitor changes in tertiary structure, and the absorbance at 597 nm and the 255-nm CD transition to monitor changes in the copper center. Reduced PC ( T m = 71 ° C) was found to be more stable than the oxidized form ( T m = 61 ° C). The T m was increased by addition of reductants, removal of oxygen, or addition of EDTA. Two distinct denatured forms (designated D 1 and D 2) were separated by anion exchange fast protein liquid chromatography. Neither form contained a native copper center. Form D 2 retained the characteristic 280-nm CD band but showed an altered far-uv CD spectrum. Its formation was inhibited by the addition of reductants or the removal of oxygen. It could be refolded to form native, Cu-PC upon incubation with copper plus a reductant such as dithionite. These results suggest that its formation involves the reversible oxidation of a group on the PC molecule, possibly a ligand to the copper such as Cys 84 or Met 92. Form D 1 occurred in the presence of ferricyanide or at high temperatures in the presence of oxygen. EDTA inhibited its formation. Form D 1 lost the 280-nm CD transition and its far-uv CD spectrum was altered. No renaturation was observed suggesting that Form D 1 is the product of an irreversible oxidation step possibly involving a histidine ligand to the copper. Forms D 1 and D 2 are not interconvertible and represent the endpoints of two different denaturation pathways.
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