Electron Paramagnetic Resonance and Circular Dichroism Studies on Milk Xanthine Oxidase

The g = 1.94 electron paramagnetic resonance signal of xanthine oxidase intensifies rapidly below 77° K and is found to resemble markedly that of spinach ferredoxin. At very low temperatures a new resonance is observed in the reduced protein at g = 2.11 and tentatively ascribed to a second type of iron chromophore. The time dependence of flavin and molybdenum during titration with substrate is complex and substantially confirms the earlier results of Bray. The iron signal does not exhibit this peculiar behavior. The development of the g = 1.94 iron signal on titration with reductants shows that maximum signal intensity is achieved with approximately 3 to 3.5 moles of xanthine and 4 moles of dithionite, in reasonable agreement with the spectrophotometric experiments reported earlier. At maximum development we account for approximately 2 electrons in the g = 1.94 and g = 2.11 signals at low temperatures. This value decreases to 1.1 electrons as the temperature is raised and the contribution of the g = 2.11 species disappears. On reduction with substrate, approximately half of the maximum intensity of these signals is attained very rapidly, the remaining development being slow. A measurement of the line width of the flavin radical yields a value of 19.4 gauss, strongly suggesting that the flavin is the blue or neutral species. The circular dichroism spectra of oxidized and reduced enzyme are recorded and the many similarities to simple non-heme iron proteins like spinach ferredoxin and adrenodoxin are discussed. Reaction with xanthine and glycolaldehyde under anaerobic conditions results in approximately half of the total circular dichroism change occurring very rapidly, the remaining changes being slow. A kinetic study of the reaction of xanthine oxidase in solution with the reducing substrate o-phenanthroline reveals that (a) the initial increase in flavin radical parallels the increase in absorbance at 620 mµ, thus supporting the identification of the flavin as the neutral species and (b) similar kinetic data are observed for the flavin and molybdenum (electron paramagnetic resonance) and the iron (absorbance, circular dichroism).