ON THE ROLE OF THE ACIDIC CLUSTER GLU-92-94 OF SPINACH FERREDOXIN-I

The role of the acidic cluster Glu 92-94 of spinach ferredoxin I in th e interaction both with the photosystem I multisubunit complex and the ferredoxin-NADP(+) reductase, either membrane-bound or purified, was studied by kinetic characterization of site-directed mutants. Three mu tants of ferredoxin have been produced to evaluate the effects of elim ination of one or two negative charges in the three specific positions of the acidic cluster. Kinetic characterization of the ferredoxin mut ants E92A/E93A, E93A and E93A/E94A as electron carriers in the photosy nthetic electron transport chain, allowed to establish that the two la tter mutants were nearly indistinguishable from the wild-type protein in their ability to be photoreduced by photosystem I and as electron d onor to the reductase in the NADP(+) photoreduction with thylakoid mem branes. The E92A/E93A ferredoxin mutant behaved very similarly to E92 mutants previously characterized. Thus, the elimination of the carboxy l groups adjacent to residue 92 did not further impaired ferredoxin I main function, i.e., as an electron carrier in NADP(+) photoreduction. The two double mutants showed a reduced rate in the cross-linking of ferredoxin to the reductase promoted by a soluble carbodiimide, indica ting an involvement of the acidic cluster in the formation of the acti ve covalent complex between the two proteins. (C) 1997 Elsevier Scienc e B.V.