title: A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.
abstract: The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.
authors.affiliation: Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Deng
authors.forename: A
authors.initials: A
authors.lastname: Aliverti
authors.forename: G
authors.initials: G
authors.lastname: Zanetti
authors.forename: A K
authors.initials: AK
authors.lastname: Arakaki
authors.forename: J
authors.initials: J
authors.lastname: Ottado
authors.forename: E G
authors.initials: EG
authors.lastname: Orellano
authors.forename: N B
authors.initials: NB
authors.lastname: Calcaterra
authors.forename: E A
authors.initials: EA
authors.lastname: Ceccarelli
authors.forename: N
authors.initials: N
authors.lastname: Carrillo
authors.forename: P A
authors.initials: PA
authors.lastname: Karplus
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D013057:Spectrum Analysis; D013816:Thermodynamics; D014443:Tyrosine / Q000235:genetics
chemical_list: D008024:Ligands; D014443:Tyrosine; D009249:NADP; D005287:Ferredoxin-NADP Reductase