Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin

We have found that the binding of NADP(+) (K-d = 0.86+/-0.11 muM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (T R, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine d inucleotide phosphate (AADP(+)) (K-d < 0.1 <mu>M) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The K -d of TR-NADP(+) and TR-AADP(+) complexes did not change in the presence of 45 muM 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 th e vicinity of the catalytic disulfide increase the content of fluorescent F R conformer (NADP(H)-binding site adjacent to flavin). The different effect s of NADP(+) and AADP(+) on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluoresc ent FR conformer upon binding of NADP(+) or AADP(+); ii) quenching of FAD f luorescence by electron-donating 3-aminopyridinium ring of AADP(+).