Reconstitution of Escherichia coli thioredoxin reductase with 1-deazaFAD. Evidence for 1-deazaFAD C-4a adduct formation linked to the ionization of an active site base.
The flavin prosthetic group (FAD) of thioredoxin reductase has been replaced by 1-deazaFAD (carbon substituted for nitrogen at position 1). Reduction of 1-deazaFAD-thioredoxin reductase by four electrons proceeds in two stages having midpoint potentials that are separated by 0.063 V. Two-electron reduced 1-deazaFAD-thioredoxin reductase (EH2) has spectral characteristics that are different from both the fully oxidized and fully reduced enzyme. The fluorescence of the 2-electron reduced enzyme shows a mixture of two EH2 species. The spectrum of one EH2 species has a single absorption peak (lambda max, 414 nm; epsilon 414, 8750 M-1 cm-1) which is similar to the spectrum of 1-deazaFAD-C-4a adducts (referred to as the 414-nm absorbing species). In the other EH2 species the electrons are in the dithiol, and it has an oxidized 1-deazaFAD spectrum (referred to as the 550-nm EH2 species). The equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is pH dependent, forming more of the 414-nm absorbing species as the pH is lowered. The pH dependence suggests the presence of an active center base having a pK of 7.41 on the 414-nm EH2 species and a thiol of pK 6.73 on the 550-nm EH2 species. These pK values are similar to the pK values determined for native enzyme having a disulfide or a dithiol (7.59 and 6.98, respectively). Thus, the pH dependence of the equilibrium between the two EH2 species of 1-deazaFAD-thioredoxin reductase is further evidence for an active site base with an ionization behavior that is linked to the chemical state of the active site disulfide moiety. The nature of the linked ionization is consistent with a thiol base ion pair formed upon disulfide reduction.
D001665:Binding Sites; D004227:Dithionite / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*
D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; C023406:1-deaza-FAD; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase