ABSTRACT
Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH at the end of the photosynthetic electron transfer chain. We previously showed that the interaction between FNR and Fd was weakened by the allosteric binding of NADP(H) on FNR, which was considered as a part of negative cooperativity. In this study, we investigated the molecular mechanism of this phenomenon using maize (Zea mays L.) FNR and Fd, as the 3D structure of this Fd:FNR complex is available. Site-specific mutants of several amino acid residues on the Fd:FNR interface were analysed for the effect on the negative cooperativity, by kinetic analysis of Fd:FNR electron transfer activity and by Fd-affinity chromatography. Mutations of Fd Arg40Gln and FNR Glu154Gln that disrupt one of the salt bridges in the Fd:FNR complex suppressed the negative cooperativity, indicating the involvement of the ion pair of Fd Arg40 and FNR Glu154 in the mechanism of the negative cooperativity. Unexpectedly, either mutation of Fd Arg40Gln or FNR Glu154Gln tends to increase the affinity between Fd and FNR, suggesting the role of this ion pair in the regulation of the Fd:FNR affinity by NADPH, rather than the stabilization of the Fd:FNR complex.