High‐resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily. (29th August 2017)

• Record Type: Journal Article
• Authors: Kean, Kelsey M.; Carpenter, Russell A.; Pandini, Vittorio; Zanetti, Giuliana; Hall, Andrea R.; Faber, Rick; Aliverti, Alessandro; Karplus, P. Andrew
• Is Part Of: FEBS journal. Volume 284:Number 19(2017)
• Page Start: 3302
• Page End: 3319
• Publication Date: 2017-08-29
• Subjects: enzyme mechanism -- flavoenzyme -- hydride transfer -- NADPH oxidase -- protein crystallography; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.14190
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Ferredoxin: NADP + reductase (FNR) is an FAD‐containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP + to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyse similar FAD‐enabled electron transfers between NAD(P)H and one‐electron carriers. Here, we define further mechanistic details of the NAD(P)H ⇌ FAD hydride‐transfer step of the reaction based on spectroscopic studies and high‐resolution (~ 1.5 Å) crystallographic views of the nicotinamide–flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP +, or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favour catalysis. Furthermore, anisotropic B‐factors show that the mobility of the C4 atom of the nicotinamide in the FNR:NADP + complex has a directionality matching that expected for boat‐like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure‐function relations for the membrane‐bound NOX enzymes for which little structural information has been available. Databases: Structural data are available in the PDB database under the accession numbers3LO8 (wild‐type), 5VW4 [Y316S:nicotinamide (P32 21)], 5VW9 [Y316S:nicotinamide (P31 21)], 5VW3 [Y316S:NADP + (P32 21)], 5VW8 [Y316S:NADP + (P31 21)], 5VW2 [Y316S:NADPH (P32 21)], 5VW5 [Y316A:nicotinamide (P32 21)], 5VW6 [Y316A:NADP + (P32 21)], 5VW7 [Y316A:NADPH (P32 21)], 5VWA [Y316F (P32 21)], and5VWB [Y316F:NADP + (P31 21)]. Enzyme Commission number: ferredoxin:NADP + reductase –E C1.18.1.2 . Abstract : Ferredoxin‐NADP + reductases (FNRs) catalyse hydride transfers between NADP(H) and ferredoxin. Combined spectroscopic studies and high‐resolution crystal structures of corn root FNR with a C‐terminal aromatic placeholder residue mutated implicate active site compression – as evidenced by mobility changes, flavin distortion and short interaction distances – as a key factor promoting hydride transfer in FNRs and the broader FNR superfamily.