SITE-DIRECTED MUTAGENESIS OF RECOMBINANT SULFITE OXIDASE - IDENTIFICATION OF CYSTEINE-207 AS A LIGAND OF MOLYBDENUM

Each of the four cysteines in rat sulfite oxidase was altered by site directed mutagenesis to serine, and the mutant proteins were expressed in Escherichia coli. Three of the replacements proved to be silent mu tations, while a single cysteine, Cys-207, was found to be essential f or enzyme activity. The C207S mutation was also generated in cloned hu man sulfite oxidase. The mutant human enzyme also displayed severely a ttenuated activity but was expressed at higher levels allowing purific a tion and spectroscopic analysis. The absorption spectrum of the isol ated molybdenum domain of the human C207S mutant displayed marked atte nuation of the peak at 350 nm and a lesser decrease in absorbance from 450-600 nm as compared with the native human molybdenum domain. The m olybdenum and molybdopterin contents of the two samples were comparabl e. These data suggest that the major features in the absorption spectr um of the native molybdenum domain arise from the binding of Cys-207 t o the molybdenum and indicate that this residue functions as a ligand of the metal.