Mechanistic insight into the initiation step of the reaction of Burkholderia pseudomallei catalase-peroxidase with peroxyacetic acid.
The reaction of the catalase-peroxidase of Burkholderia pseudomallei with peroxyacetic acid has been analyzed using stopped-flow spectrophotometry. Two well-defined species were observed, the first defined by an increase in intensity and narrowing of the Soret band at 407 nm and a 10-nm shift of the charge transfer band from 635 to 625 nm. These features are consistent with a ferric spectrum with a greater proportion of sixth-coordination character and are assigned to an Fe(III)-peroxyacetic acid complex. Complementary 9-GHz EPR characterization of the changes in the ferric signal of the resting enzyme induced by the binding of acetate in the heme pocket substantiates the proposal. Kinetic analysis of the spectral changes as a function of peroxyacetic acid concentration revealed two independent peroxyacetic acid binding events, one coincident with formation of the Fe(III)-peroxyacetic acid complex and the other coincident with the heme oxidation to the subsequent ferryl intermediate. A model to explain the need for two peroxyacetic acid binding events is proposed. The reaction of the W330F variant followed similar kinetics, although the characteristic spectral features of the Fe(IV)=O Por(*+) species were detected. The variant D141A lacking an aspartate at the entrance to the heme cavity as well as the R108A and D141A/R108A variants showed no evidence for the Fe(III)-peroxyacetic acid complex, only the formation of ferryl species with absorbance maxima at 414, 545, and 585 nm.
Wiseman
Ben
B
Department of Microbiology, University of Manitoba, Winnipeg, MB, Canada.
Colin
Julie
J
Smith
Andrew T
AT
Ivancich
Anabella
A
Loewen
Peter C
PC
D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D016957:Burkholderia pseudomallei / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D050505:Mutant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010463:Peracetic Acid / Q000032:analysis* / Q000378:metabolism*; D010544:Peroxidases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry
D001426:Bacterial Proteins; D050505:Mutant Proteins; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; D010463:Peracetic Acid