Charge reversal mutations in a conserved acidic patch in Anabaena ferredoxin can attenuate or enhance electron transfer to ferredoxin:NADP+ reductase by altering protein/protein orientation within the intermediate complex.

A series of charge reversal mutations in a highly conserved acidic patch on the surface of Anabaena ferredoxin (Fd), comprising residues D67, D68, and D69, have been constructed by site-directed mutagenesis. One such mutant, D68K, has a rate constant for electron transfer (et) to Anabaena ferredoxin:NADP+ reductase (FNR) at low ionic strength (I = 12 mM) which is 2.5 times larger than wild type (9000 vs 3600 s-1). This mutant Fd became indistinguishable from the wild-type protein in its reactivity at I > or = 100 mM. The other mutants showed various degrees of impairment in their et reactions with FNR over the entire range of ionic strengths. The degrees of such impairment for the D67K and D69K mutants were similar to that of the double mutant D67K/D69K. The double mutant D68K/ D69K had et activity intermediate between these mutants and wild type, whereas incorporation of the "super" mutation, D68K, into the double mutant, resulting in the D67K/D68K/D69K triple mutant, did not significantly alter the impairment caused by the D67K/D69K double mutation. Binding constants for complex formation (Kd) between the oxidized mutant proteins and oxidized FNR (except for that of the triple mutant which was not measurable), and the kinetically determined Kd values for the intermediate Fdred:FNRox complex, showed no correlation with et rate constants or with the extent of charge reversal. These results indicate that hydrophobic interactions play a key role in determining complex stability. They also provide strong support for the contention that the specific protein/protein geometry within the Fdred:FNRox intermediate complex is the major determinant of the et rate constants in this series of mutants, and that this is optimized largely by hydrophobic rather than electrostatic interactions. When electrostatic forces are dominant, as they are at low ionic strength, this can lead to nonoptimal et orientations.