Steady state and picosecond time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase

Steady state and time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase (XO) have been carried out to investigate the con formational changes associated with the replacement of the molybdenum doubl e bonded sulphur by oxygen and the removal of the flavin adenine dinucleoti de (FAD). The steady state quenching experiments of the intrinsic tryptopha n residues of the enzyme show that all the nine tryptophans are accessible to neutral quencher, acrylamide, in the native as well as desulpho and defl avo enzymes. However, the number of the tryptophan residues accessible to t he ionic quenchers, potassium iodide and cesium chloride, increases upon re moval of the FAD centre from the enzyme. This indicates that two tryptophan residues move out from the core of the enzyme to the solvent upon the remo val of the FAD. The time-resolved fluorescence studies were carried out on the native, desulpho and deflavo XO by means of the time-correlated single photon counting technique, and the data were analysed by discrete exponenti al and maximum entropy methods. The results show that the fluorescence deca y curve fitted best to a three-exponential model with lifetimes tau(1) = 0. 4, tau(2) = 1.4 and tau(3) = 3.0 ns for the native and desulpho XO, and tau (1) = 0.7, tau(2) = 1.7 and tau(3) = 4.8 ns for the deflavo XO. The replace ment of the molybdenum double bonded sulphur by oxygen in the desulpho enzy me does not cause any significant change of the lifetime components. Howeve r, removal of the FAD centre causes a significant change in the shortest an d longest lifetime components indicating a conformational change in the def lavo XO possibly in the flavin domain. Decay-associated emission spectra at various emission wavelengths have been used to determine the origin of the lifetimes. The results show that tau(1) and tau(3) Of the native and desul pho XO originate from the tryptophan residues which are completely or parti ally accessible to the solvent but tau(2) corresponds to those residues whi ch are buried in the core of the enzyme and not exposed to the solvent. For deflavo enzyme, tau(2) is red shifted compared to the native enzyme indica ting the movement of tryptophan residues from the core of the enzyme to the solvents. (C) 2000 Elsevier Science B.V. All rights reserved.