Spectroscopic and kinetic characterization of the spinach plastocyanin mutant Tyr83-His: a histidine residue with a high p K value

Abstract

Tyrosine-83 in spinach plastocyanin (Pc) has been modified by site-directed mutagenesis to a histidine. An NMR titration yields a p K value of 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 35 mV at pH 7.5, but only slightly, if at all, at pH 8.9. EPR and optical absorption bands associated with the copper site are not affected by the mutation, either at pH 7.5 or at pH 8.9. The electron transfer (ET) to Photosystem I (PS I), as studied by a flash-photolysis technique, 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 interpreted with a model that includes a rate-limiting conformational change in the Pc-PS I complex which precedes the intracomplex ET (Bottin, H. and Mathis, P. (1985) Biochemistry 24, 6453–6460). The slower kinetics at the lower pH for the mutant is attributed to a dual effect of the protonation 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 residue is not a part of the ET pathway to PS I.