SPECTROSCOPIC AND KINETIC CHARACTERIZATION OF THE SPINACH PLASTOCYANIN MUTANT TYR83-HIS - A HISTIDINE RESIDUE WITH A HIGH PK VALUE

Tyrosine-83 in spinach plastocyanin (Pc) has been modified by site-dir ected mutagenesis to a histidine. An NMR titration yields a pK value o f 8.44 for this residue. The high value is probably due to the acidic residues close to this site. The reduction potential is increased by 3 5 mV at pH 7.5, but only slightly, if at all, at pH 8.9. EPR and optic al absorption bands associated with the copper site are not affected b y the mutation, either at pK 7.5 or at pH 8.9. The electron transfer ( ET) to Photosystem I (PS I), as studied by a flash-photolysis techniqu e, is pH dependent for the mutant, being slower than the wild type at pH 7.5 but more similar to it at pH 8.9. The data have been interprete d with a model that includes a rate-limiting conformational change in the Pc-PS I complex which precedes the intracomplex ET (Bottin, H. and Maths, P. (1985) Biochemistry 24, 6453-6460). The slower kinetics at the lower pH for the mutant is attributed to a dual effect of the prot onation of the His-83 residue: (i) A destabilization of the ''close'' bound conformation, i.e., the one competent in electron transfer, and (ii) a smaller intracomplex ET rate constant, partly due to a smaller driving force for ET. From this it is concluded that the Tyr-83 residu e is not a part of the ET pathway to PS I.