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Abstract The rapid anaerobic reduction of xanthine oxidase by substrate has been studied using rapid freeze electron paramagnetic resonance, stopped flow spectrophotometry, and steady state kinetic analyses. Four major points have been resolved. The two types of molybdenum EPR signals which appear in the rapid phases of reduction are interconvertible by a prototopic equilibrium. The rapid optical bleaching which occurs during reduction has identical kinetics to the appearance of the non-heme iron-sulfur group signals determined by rapid freeze EPR studies. This rate of reduction is identical to the Vmax determined by steady state kinetic analysis implying that the oxygen independent steps must be rate limiting during catalysis. There are significant deuterium kinetic isotope effects on all of the EPR-detectable species as well as specific deuterium effects on the line shape of the molybdenum EPR spectrum. These are strong pieces of evidence that substrates transfer a hydride ion to molybdenum in the course of reducing the enzyme.
Edmondson et al. (Sat,) studied this question.