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 conformational changes associated with the replacement of the molybdenum double bonded sulphur by oxygen and the removal of the flavin adenine dinucleotide (FAD). The steady state quenching experiments of the intrinsic tryptophan residues of the enzyme show that all the nine tryptophans are accessible to neutral quencher, acrylamide, in the native as well as desulpho and deflavo enzymes. However, the number of the tryptophan residues accessible to the ionic quenchers, potassium iodide and cesium chloride, increases upon removal 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 removal 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 exponential and maximum entropy methods. The results show that the fluorescence decay 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 replacement of the molybdenum double bonded sulphur by oxygen in the desulpho enzyme does not cause any significant change of the lifetime components. However, removal of the FAD centre causes a significant change in the shortest and longest lifetime components indicating a conformational change in the deflavo 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 desulpho XO originate from the tryptophan residues which are completely or partially accessible to the solvent but tau(2) corresponds to those residues which 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 indicating the movement of tryptophan residues from the core of the enzyme to the solvents.
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Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, 400 005, Mumbai, India. apurba@mail.utexas.edu
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D020106:Acrylamide; D002586:Cesium; D002712:Chlorides; D011193:Potassium Iodide; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry*
D002712:Chlorides; D011193:Potassium Iodide; D002586:Cesium; D020106:Acrylamide; D014364:Tryptophan; D014969:Xanthine Oxidase; C028019:cesium chloride