THE INVOLVEMENT OF THE 2 ACIDIC PATCHES OF SPINACH PLASTOCYANIN IN THE REACTION WITH PHOTOSYSTEM-I

Six different spinach plastocyanin mutants have been constructed by si te-directed mutagenesis and expressed in Escherichia coli to probe the importance of the two acidic patches in the interaction with photosys tem I. The mutants were: Asp42Lys, Glu43Asn, Glu43Lys, Glu43Gln/Asp44A sn, Glu59Lys/Glu60Gln and Glu43Asn/Glu59Lys/Glu60Gln and they have bee n characterised by optical absorption and EPR spectroscopy, redox titr ations and isoelectric focusing. The electron transfer to photosystem I was investigated by flash-induced time-resolved absorption measureme nts at 830 nm. The kinetics were interpreted with a model that incorpo rates a rate-limiting conformational change from inactive to active fo rms of the plastocyanin-photosystem I complex. AU mutations resulted i n a displacement of the equilibrium towards the inactive conformation. The strongest impairment of the electron transfer was found for mutat ions in the larger acidic patch, in particular upon modification of re sidues 43 or 44. However, mutations of residues 59 and 60 in the small er acidic patch also resulted in a lower reactivity. (C) 1997 Elsevier Science B.V.