High-resolution studies of hydride transfer in the ferredoxin:NADP+ reductase superfamily

FEBS J. 2017 Oct;284(19):3302-3319. doi: 10.1111/febs.14190. Epub 2017 Aug 29.

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 numbers 3LO8 (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)], and 5VWB [Y316F:NADP+ (P31 21)]. Enzyme Commission number: ferredoxin:NADP+ reductase - E C1.18.1.2.

Keywords: NADPH oxidase; enzyme mechanism; flavoenzyme; hydride transfer; protein crystallography.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Biocatalysis
  • Catalytic Domain
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Ferredoxin-NADP Reductase / chemistry*
  • Ferredoxin-NADP Reductase / genetics
  • Ferredoxin-NADP Reductase / metabolism
  • Flavin-Adenine Dinucleotide / chemistry*
  • Flavin-Adenine Dinucleotide / metabolism
  • Gene Expression
  • Humans
  • Kinetics
  • Models, Molecular
  • NAD / chemistry*
  • NAD / metabolism
  • NADP / chemistry*
  • NADP / metabolism
  • NADPH Oxidases / chemistry*
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Plant Roots / chemistry
  • Plant Roots / enzymology
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Zea mays / chemistry
  • Zea mays / enzymology

Substances

  • Plant Proteins
  • Recombinant Proteins
  • NAD
  • Flavin-Adenine Dinucleotide
  • NADP
  • Ferredoxin-NADP Reductase
  • NADPH Oxidases

Associated data

  • figshare/10.6084/m9.figshare.5328784