Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin.
We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.
Nivinskas
H
H
Institute of Biochemistry, Vilnius, Lithuania. ncenas@bchi.lt
Jacquot
J P
JP
Cenas
N
N
D000227:Adenine Nucleotides / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D011487:Protein Conformation; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D000227:Adenine Nucleotides; D005182:Flavin-Adenine Dinucleotide; D013879:Thioredoxins; D009249:NADP; C039995:3-aminopyridine-1,N(6)-ethenoadenine dinucleotide phosphate; D013880:Thioredoxin-Disulfide Reductase