The involvement of the two acidic patches of spinach plastocyanin in the reaction with photosystem I.

Six different spinach plastocyanin mutants have been constructed by site-directed mutagenesis and expressed in Escherichia coli to probe the importance of the two acidic patches in the interaction with photosystem I. The mutants were: Asp42Lys, Glu43Asn, Glu43Lys, Glu43Gln/Asp44Asn, Glu59Lys/Glu60Gln and Glu43Asn/Glu59Lys/Glu60Gln and they have been characterised by optical absorption and EPR spectroscopy, redox titrations and isoelectric focusing. The electron transfer to photosystem I was investigated by flash-induced time-resolved absorption measurements at 830 nm. The kinetics were interpreted with a model that incorporates a rate-limiting conformational change from inactive to active forms of the plastocyanin-photosystem I complex. All mutations resulted in a displacement of the equilibrium towards the inactive conformation. The strongest impairment of the electron transfer was found for mutations in the larger acidic patch, in particular upon modification of residues 43 or 44. However, mutations of residues 59 and 60 in the smaller acidic patch also resulted in a lower reactivity.