Structure-function correlations for the [2Fe-2S] ferredoxin-ferredoxin: NADP+ reductase system in anabaena

Studies were conducted to test the role of the prolein matrix in medial ing electron transfer between the vegetative cell [2Fe-2S] ferredoxin (VFd) and ferredoxin:NADP+ reductase (FNR) from Anabacna. Twelve site-directed VKd mutants were prepared. E94K, E94Q, F65A, F65I. and S47A exhibited negli gibîe kinetic activity with FNR. Their reduction potentials were shifted 47 1<> 0;i mV positively compared to wild-type {E - II84 mV versus S.H.K., pH 7..">i, thus lowering the driving force of electron transfer to F\R (L," -'J2't mV. pH 7.n); but in electrostatic complexes of E94K, F65I. or S47A with tNK. electron transfer becomes isopotential or thermodynamic.ally favorable, largelv due io positive potential shifts in the flavin of FNR. The x-ray crystal siruc lures of F94K and S47A revealed no significant structural changes near th"' [2Fe-'2SJ cluster and only small changes in Co backbone positions which could not be correlated with their functional properties. These data suggest that the impaired kinetic reactivity of these mutants is due to changes in the mutual ori filiation of Fd and FNR in the transient protein-protein complex. Specifically, a hydrogen bond between the side chains of S47 and F9-1 and aromaticily al lesidue fi") are required for efficient electron transfer and a réduction potential ah negative as wild type.