Abstract
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.
Highlights
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
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
In order to investigate the molecular mechanism of this phenomenon, here we studied the influence of NADP+ and NADPH on the binding between Fd and FNR by different methods (Fd-affinity chromatography, and kinetic and calorimetric analyses) using maize recombinant proteins with known steric structures of FNR and Fd : FNR complex [1], and the effects of sitedirected mutations at the sites involving the binding of NADP(H) on FNR were investigated
Summary
Cloning and preparation of maize leaf FNR (L-FNR) [13] and maize leaf Fd I [14,15] were described previously. The activity of NADPHdependent electron transfer from FNR to Fd was measured using cytochrome c (cyt c) as a final electron acceptor basically as described previously [13] in the reaction mixtures containing different concentrations of NADPH. Calorimetric experiments were performed basically as described previously [17] except that NaCl was omitted in the reaction (50 mM Tris/HCl, pH 7.5). Calorimetric experiments were performed with an Auto-iTC 200 instrument (GE Healthcare Biosciences, Chicago, IL, USA) at 298 K. 500 lM Fd I was titrated into 50 lM wild-type or mutant L-FNR in the ITC cell. The injection volume was 1.0 lL, and the cell was continuously stirred at 1000 r.p.m. Thermodynamic parameters of the complex formation between Fd and FNR were obtained as described previously [18] using the one set of independent binding site model supplied by the MICROCAL ORIGIN 7.0.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
