FIGURE 4. Mutation of Asp 32 or Ser 167 eliminates DMB production.

HPLC traces (280 nm) of reactions performed with BluB proteins containing the point mutations Asp32Asn or Ser167Gly. The peaks corresponding to FMN and DMB, as confirmed by retention time and ultraviolet/visible spectra, are labelled. No DMB is detected in the Asp32Asn mutant; DMB produced by the Ser167Gly mutant is 3% of the wild-type level. No DMB is detected in reactions containing BluB with Asp32Ala, Asp32Ser, or Ser167Cys point mutations (not shown). The size of the small peaks at 22.7 and 25.8 min in the reactions with mutant enzymes is reduced by addition of catalase, indicating that H₂O₂ is generated in reactions with mutant BluB, probably via futile cycling through a peroxyflavin intermediate (not shown).