The Role of Tyrosine 343 in Substrate Binding and Catalysis by Human Sulfite Oxidase

Abstract

In the crystal structure of chicken sulfite oxidase, the residue Tyr 322 (Tyr343 in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, kcat was somewhat impaired (∼34% wild-type activity at pH 8.5), whereas the Kmsulfite showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, kredheme changed very little over the entire pH range, with a significant increase in Kdsulfite at high pH. The k redheme of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k redheme and Kdsulfite were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (kredMo = 972 s-1 at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr343 residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.