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Ferredoxin:NADPH oxidoreductase is recruited to thylakoids by binding to a polyproline type II helix in a pH-dependent manner - PubMed

  • ️Fri Jan 01 2010

Ferredoxin:NADPH oxidoreductase is recruited to thylakoids by binding to a polyproline type II helix in a pH-dependent manner

Ferdinand Alte et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Ferredoxin:NADPH oxidoreductase (FNR) is a key enzyme of photosynthetic electron transport required for generation of reduction equivalents. Recently, two proteins were found to be involved in membrane-anchoring of FNR by specific interaction via a conserved Ser/Pro-rich motif: Tic62 and Trol. Our crystallographic study reveals that the FNR-binding motif, which forms a polyproline type II helix, induces self-assembly of two FNR monomers into a back-to-back dimer. Because binding occurs opposite to the FNR active sites, its activity is not affected by the interaction. Surface plasmon resonance analyses disclose a high affinity of FNR to the binding motif, which is strongly increased under acidic conditions. The pH of the chloroplast stroma changes dependent on the light conditions from neutral to slightly acidic in complete darkness or to alkaline at saturating light conditions. Recruiting of FNR to the thylakoids could therefore represent a regulatory mechanism to adapt FNR availability/activity to photosynthetic electron flow.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.

Structures of the FNR:Tic62-peptide complex using Molscript (27) and Bobscript (28). (A) Stereo view of the two FNR-subunits, shown as gray ribbon plots and denoted as FNR A and FNR B, respectively. The Tic62 peptide, colored in green, and both FAD cofactors, colored in yellow, are shown as ball-and-stick models. N and C termini of the peptide are numbered according to the full-length Tic62 protein. (B) Stereo view of the 2Fo-Fc electron density map (countered at 1σ) for the Tic62 peptide in the complex interface, in which the Tic62 peptide has been omitted for phasing. FNRs are represented in coils and amino acid residues of both FNR subunits complexing the Tic62 peptide are depicted as sticks. Hydrogen bonds and a salt bridge are indicated by black dashed lines. Here, the peptide amino acids are numbered from 1 to 27. (see also

Table S1

). (C) Alignment of the C termini of Tic62/Trol proteins from different vascular plants containing the FNR-MRM. The red frame indicates the strictly conserved core-motif. Ps, Pisum sativum: At, Arabidopsis thaliana: Os, Oryza sativa: Pt, Populus trichocarpa: OsJ, Oryza sativa subsp. japonica. The OsJ_04356 sequence is related to a putative uncharacterized protein. (see also

Fig. S1A

)

Fig. 2.
Fig. 2.

Structural overlay of the Tic62 peptide with three PPII helices. Only Pro-side chains are shown. The backbone dihedral angles of the PPII helices are φ = -75° and Ψ = 145°. The dihedral angles of the first five amino acids of the Tic62 peptide (segment 1, Lys1—Pro5) are in the respective range and the backbone widely matches the backbone of PPII helix 1. The three proline residues 8, 17, and 18 together with Tyr9 and Lys16 of the FNR-MRM form a second axis of a PPII helix (PPII helix 2; segment 2, Pro8–Pro18).The third segment (Pro21–Pro26) in the FNR-MRM is indicated by PPII helix 3.

Fig. 3.
Fig. 3.

Surface representation of the FNR:Tic62-peptide complex using GRASP software (29). Stereo view of both FNR subunits in the complex with (A) and without (B) the Tic62 peptide, which is colored in green. The FNR-MRM fills a large cavity between the two FNR-molecules, thereby significantly expanding the contact surface.

Fig. 4.
Fig. 4.

Schematic model of pH-dependent FNR-storage at the thylakoid membrane by Tic62. During dark periods, when photosynthesis is shut down, FNR strongly interacts with the Tic62 C terminus due to an acidic stromal pH. Hence, FNR is attached to the thylakoid membrane (Left), where it is stored and stabilized until reactivation of photosynthesis. Low light quantities (indicated by a small, red arrow) induce a modest photosynthetic activity, resulting in a higher stromal pH, which causes a partial release of FNR molecules into the stroma (Center). Under these conditions, possibly both FNR populations, at the membrane as well as in the stroma, contribute to the production of an adequate amount of reduction equivalents (NADPH). High light conditions (indicated by large, red arrows) evoke high photosynthetic activity, leading to an alkaline stroma, since protons are transported into the thylakoid lumen. Under these conditions, FNR is predominantly found in a Tic62-free form, mediating the transfer of electrons from ferredoxin (Fd) to NADP+ (Right). For the sake of clarity, the role of Tic62 in the chloroplast redox shuttling system is not depicted.

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