Highly nonproductive complexes with anabaena ferredoxin at low ionic strength are induced by nonconservative amino acid substitutions at Glu139 in Anabaena ferredoxin : NADP(+) reductase

Ferredoxin (Fd) and ferredoxin:NADP(+) reductase (FNR) from Anabaena functi on in photosynthetic electron transfer (et). The et interaction between the FNR charge-reversal mutant E139K and Fd at 12 mM ionic strength (mu) is ex tremely impaired relative to the reaction with wt FNR, and the dependency o f k(obs) on E139K concentration shows strong upward curvature at protein co ncentrations greater than or equal to 10 muM. However, at values of mu grea ter than or equal to 200 mM, reaction rates approach those of wild-type FNR , and normal saturation kinetics are observed. For the E139Q mutant, which is also significantly impaired in its et interaction with Fd at low FNR con centrations and low mu values, the dependency of k(obs) on E139Q concentrat ion shows a smaller degree of upward curvature at mu = 12 and 100 mM and sh ows saturation kinetics at higher values of mu. wt FNR and the E139D mutant both show a slight amount of upward curvature at FNR concentrations >30 mu M at mu = 12 mM but show the expected saturation kinetics at higher values of mu. These results are expained by a mechanism in which the mutual orient ation of the proteins in the complex formed at low ionic strength with the E139K mutant is so far from optimal that it is almost unreactive. At increa sed E139K concentrations, the added mutant FNR reacts via a collisional int eraction with the reduced Fd present in the unreactive complex. The et reac tivity of the low ionic strength complexes depends on the particular amino acid substitution, which via electrostatic interactions alters the specific geometry of the interface between the two proteins. The presence of a nega tive charge at position 139 of FNR allows the most optimal orientations for et at ionic strengths below 200 mM.