Chemical modification of spinach plastocyanin: separation and characterization of four different forms

Spinach plastocyanin has been chemically modified by using a water-soluble carbodiimide to form an amide bond between a protein carboxyl group and one amino group of ethylenediamine. This reaction produced four distinct species of chemically modified plastocyanin which could be separated either by polyacrylamide gel electrophoresis (in the absence of sodium dodecyl sulfate) or by ion-exchange chromatography on DEAE-Sephadex. The four forms contained 2.1, 3.2, 4.1, and 6.3 mol of ethylenediamine per mol of plastocyanin and were labeled fractions IV, III, II, and I, respectively. All four fractions showed a shift in midpoint redox potential of greater than +40 mV. In contrast, the Km for P700+ reduction decreased with increasing extents of modification. The addition of Mg2+ ions decreased the Km for the negatively charged fractions II, III, and IV but increased it for the positively charged fraction I. Fraction III showed an increased incorporation of ethylenediamine into residues 42-45 which are located approximately 18 A distant from the copper. This fraction also showed a maximal increase in redox potential. Potential calculations showed that the electrostatic effect of incorporating two positive charges at this distance was sufficient to produce a shift in redox potential of +40 mV. However, an electrostatically induced conformational change which would affect the environment of the copper cannot be ruled out.