Effect of carboxyl group modification on redox properties and electron donation capability of spinach plastocyanin.
Spinach plastocyanin was chemically modified by using a water-soluble carbodiimide to form an amide bond between a protein carboxyl group and one amino group of ethylenediamine. On the average, four plastocyanin carboxyl groups were replaced with positively charged amino groups. Modified plastocyanin facilitated high rates of electron donation to the oxidized P700 reaction center of photosystem I particles in the absence of cations. Control plastocyanin was totally inactive in the absence of divalent cations due to charge repulsion between the negatively charged plastocyanin and photosystem I proteins. The Km for the binding of modified plastocyanin to photosystem I particles was 2.1 uM compared to 36.5 uM for control plastocyanin in the presence of MgCl2. Therefore, chemical modification was mre effective than charge shielding by cations in facilitating the binding of plastocyanin to photosystem I. Chemical modification also increased the midpoint redox potential of plastocyanin from +380 to +420 mV, which indicated an alteration of the copper environment. Ethylenediamine was covalently attached to tryptic peptides from plastocyanin that contained amino acid residues 42-45, 59-61, and 68 which are highly conserved glutamic acid and aspartic acid residues. Chemically altering the charge on these residues changed the activity of plastocyanin which indicates that these amino acids are involved in the ionic regulation of the plastocyanin-PSI interaction. Because chemical modification also altered the environment of the chromophore, certain of these amino acids must be located near the copper site.
D002234:Carbodiimides / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004579:Electron Transport; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000378:metabolism*; D010944:Plants / Q000378:metabolism*; D010970:Plastocyanin / Q000378:metabolism*
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D010940:Plant Proteins; D015636:Magnesium Chloride; D010970:Plastocyanin; D008274:Magnesium