Molecular mechanism of negative cooperativity of ferredoxin-NADP+ reductase by ferredoxin and NADP(H): involvement of a salt bridge between Asp60 of ferredoxin and Lys33 of FNR.

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 FNR and Fd, as the three-dimensional structure of this Fd:FNR complex is available. NMR chemical shift perturbation analysis identified a site (Asp60) on Fd molecule which was selectively affected by NADP(H) binding on FNR. Asp60 of Fd forms a salt bridge with Lys33 of FNR in the complex. Site-specific mutants of FdD60 and FNRK33 suppressed the negative cooperativity (downregulation of the interaction between FNR and Fd by NADPH), indicating that a salt bridge between FdD60 and FNRK33 is involved in this negative cooperativity.

NADP(H) allosterically regulates the interaction between ferredoxin and ferredoxin-NADP+ reductase.

Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) at the end of the photosynthetic electron transfer chain and converts NADP+ to NADPH. The interaction between Fd and FNR in plants was previously shown to be attenuated by NADP(H). Here, we investigated the molecular mechanism of this phenomenon using maize FNR and Fd, as the three-dimensional structure of this complex is available. NADPH, NADP+ , and 2'5'-ADP differentially affected the interaction, as revealed through kinetic and physical binding analyses. Site-directed mutations of FNR which change the affinity for NADPH altered the affinity for Fd in the opposite direction to that for NADPH. We propose that the binding of NADP(H) causes a conformational change of FNR which is transferred to the Fd-binding region through different domains of FNR, resulting in allosteric changes in the affinity for Fd.