A STD-NMR study of the interaction of the Anabaena ferredoxin-NADP+ reductase with the coenzyme

Abstract

Ferredoxin-NADP+ reductase (FNR) catalyzes the electron transfer from ferredoxin to NADP+ via its flavin FAD cofactor. To get further insights in the architecture of the transient complexes produced during the hydride transfer event between the enzyme and the NADP+ coenzyme we have applied NMR spectroscopy using Saturation Transfer Difference (STD) techniques to analyze the interaction between FNRox and the oxidized state of its NADP+ coenzyme. We have found that STD NMR, together with the use of selected mutations on FNR and of the non-FNR reacting coenzyme analogue NAD+, are appropriate tools to provide further information about the the interaction epitope.

Figure 1

Theoretical models for binary FNR:NADP⁺ and ternary Fd:FNR:NADP⁺ complexes from Anabaena. (A) Structural model at the equilibrium of the molecular dynamics simulations of a catalytically competent complex between reduced FNR and NADP⁺ [14]. (B) Structural model for the Fd:FNR:NADP⁺ ternary complex. The model has been constructed by structural alignment of the equilibrium molecular dynamics FNR:NADP⁺ complex (A) with the crystal structure of the Fd:FNR complex (PDB 1ewy) [24]. C. Detail of the relative disposition between the NADP⁺ coenzyme and the FAD cofactor in the binary FNR:NADP⁺ complex. The polypeptide chain of FNR is shown in wheat (in A and B as surface and in C as cartoon). FAD and NADP⁺ are represented in sticks with carbons in orange and blue, respectively. N4 and A1’ positions of NADP⁺ are black labeled to help orientating the molecule. In B Fd is shown in violet cartoon with its iron-sulphur cluster in spheres. Residues mutated in this work are highlighted in raspberry either as surface dots (A and B) or CPK sticks (C).

Figure 2

Formulae of NAD⁺ (R = H) and NADP⁺ (R = PO₃H⁻) and numbering used in the text.

Figure 3

Relative STD experimental for WT Anabaena FNR and NADP⁺ (dots) and theoretically predicted (lines) with CORCEMA for the crystallographic structure (PDB: 1gjr), for the adenosine (left) and for the nicotinamide (right) nucleotides of the coenzyme.

Figure 4

Relative STD experimental (dots) and theoretically predicted (lines) with CORCEMA for one structure taken from MD simulation and further minimized, for the adenosine (left) and for the nicotinamide (right) nucleotides.