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 mutations, while a single cysteine, Cys-207, was found to be essential for enzyme activity. The C207S mutation was also generated in cloned human sulfite oxidase. The mutant human enzyme also displayed severely attenuated activity but was expressed at higher levels allowing purification and spectroscopic analysis. The absorption spectrum of the isolated molybdenum domain of the human C207S mutant displayed marked attenuation 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 molybdenum and molybdopterin contents of the two samples were comparable. These data suggest that the major features in the absorption spectrum of the native molybdenum domain arise from the binding of Cys-207 to the molybdenum and indicate that this residue functions as a ligand of the metal.
Garrett
R M
RM
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Rajagopalan
K V
KV
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003545:Cysteine; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008982:Molybdenum / Q000032:analysis / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry; D013054:Spectrophotometry, Atomic; D014357:Trypsin / Q000378:metabolism
D008024:Ligands; D011994:Recombinant Proteins; D008982:Molybdenum; D050862:Oxidoreductases Acting on Sulfur Group Donors; D014357:Trypsin; D003545:Cysteine