Mechanism of coenzyme recognition and binding revealed by crystal structure analysis of ferredoxin-NADP+ reductase complexed with NADP+

The flavoenzyme ferredoxin-NADP(+) reductase (FNR) catalyses the production of NADPH in photosynthesis. The three-dimensional structure of FNR presents two distinct domains, one for binding of the FAD prosthetic group and the other for NADP(+) binding. In spite of extensive experiments and different crystallographic approaches, many aspects about how the NADP(+) substrate binds to FNR and how the hydride ion is transferred from FAD to NADP(+) remain unclear. The structure of an complex from Anabaena has been determined by X-ray diffraction analysis of the cocrystallised units to 2.1 Angstrom resolution. Structural perturbation of FNR induced by complex formation produces a narrower cavity in which the 2-phospho-AMP and pyrophosphate portions of the NADP(+) are perfectly bound. In addition, the nicotinamide mononucleotide moiety is placed in a new pocket created near the FAD cofactor with the ribose being in a tight conformation. The crystal structure of this FNR:NADP(+) complex obtained by cocrystallisation displays NADP(+) in an unusual conformation and can be considered as an intermediate state in the process of coenzyme recognition and binding. Structural analysis and comparison with previously reported complexes allow us to postulate a mechanism which would permit efficient hydride transfer to occur. Besides, this structure gives new insights into the postulated formation of the ferredoxin:FNR:NADP(+) ternary complex by prediction of new intermolecular interactions, which could only exist after FNR:NADP(+) complex formation. Finally, structural comparison with the members of the broad FNR structural family also provides an explanation for the high specificity exhibited by FNR for NADP(+)/H versns NAD(+)/H. (C) 2002 Elsevier Science Ltd. All rights reserved.