title: The effect of N-bromosuccinimide on ferredoxin:NADP+ oxidoreductase.
abstract: Treatment of spinach leaf ferredoxin:NADP+ oxidoreductase (FNR) with N-bromosuccinimide (NBS), under conditions where approximately one tryptophan residue per enzyme was modified, resulted in a loss of between 80 and 85% of the activity of the enzyme when electron transfer from NADPH to either ferredoxin or 2,6-dichlorophenol-indophenol was measured. Amino acid analysis revealed no detectable modification by NBS of any FNR amino acids other than tryptophan. Complex formation with ferredoxin, but not with NADP+, prevented both the inhibition of activity and the modification of tryptophan caused by the treatment with NBS. Modification of one FNR tryptophan residue had no significant effect on the Km values of the enzyme for either ferredoxin or NADPH or on the binding constants for the FNR complexes with either ferredoxin or NADP+. NBS treatment had only very small effects on the absorbance and circular dichroism spectra of FNR and did not significantly affect either the oxidation-reduction midpoint potential of the FAD prosthetic group of the enzyme or inhibit the reduction of the FAD group by NADPH. These results raise the possibility that a tryptophan residue may play a role in the electron transfer between the FAD of FNR and the enzyme substrate, ferredoxin.
authors.affiliation: Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061, USA.
authors.forename: M
authors.initials: M
authors.lastname: Hirasawa
authors.forename: S
authors.initials: S
authors.lastname: Kleis-SanFrancisco
authors.forename: P A
authors.initials: PA
authors.lastname: Proske
authors.forename: D B
authors.initials: DB
authors.lastname: Knaff
mesh_terms: D001974:Bromosuccinimide / Q000378:metabolism* / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D010084:Oxidation-Reduction; D018724:Spinacia oleracea / Q000201:enzymology*; D014364:Tryptophan / Q000378:metabolism
chemical_list: D014364:Tryptophan; D005287:Ferredoxin-NADP Reductase; D001974:Bromosuccinimide