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Spectroscopic and Kinetic Studies of Arabidopsis thaliana Sulfite Oxidase

  • Author(s): Byrne, Robert Stephen
  • Advisor(s): Hille, Charles R
  • et al.
Abstract

The sulfite oxidases are a family of enzymes characterized by the nature of their molybdenum centers. There are three classes of sulfite oxidizing enzymes: vertebrate sulfite oxidases, plant sulfite oxidase and bacterial sulfite dehydrogenases. All these proteins catalyze the oxidation of sulfite to sulfate - as part of sulfite detoxification in plant and animals and during chemolithotrophic growth in bacteria. While sharing a common overall reaction, each family has distinct characteristics both in the overall structure and in the reaction mechanism. Here we have set out to examine the reaction mechanism of sulfite oxidase from Arabidopsis thaliana, a protein that is unique compared to the other sulfite oxidizing enzymes. Previous work, utilizing continuous wave electron paramagnetic resonance (CW-EPR), has shown that a unique signal is seen in plant sulfite oxidase. The nature of this signal has been studied here using two different pulsed EPR techniques, two-pulse electron spin echo envelope modulation (ESEEM) and two-dimensional hyperfine sublevel correlation (2D-HYSCORE). In these studies we show that this signal is due to the formation of a sulfate-bound Mo(V) intermediate, a finding of major mechanistic implication since it implies that plant sulfite oxidases are catalytically active while previous studies have shown that vertebrate and bacterial sulfite oxidase become trapped in this form yielding a catalytic dead end. We also show here kinetic evidence of superoxide production by this enzyme during its oxidative half-reaction with oxygen, a reaction not shared with the other sulfite oxidizing enzymes. Through rapid-reaction kinetic studies, we show that this reaction involves two sequential one-electron transfers, each generating an equivalent of superoxide. Finally, we have examined the reaction of plant sulfite oxidase with dimethylsulfite and shown that a substrate lone pair attack at the equatorial oxo group of the molybdenum center must be responsible for initiation of catalysis. The interpretation of these results and their implications are discussed in the context of a modified reaction mechanism to explain the behavior of this protein during the oxidative half-reaction.

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