Total conversion of bifunctional catalase-peroxidase (KatG) to monofunctional peroxidase by exchange of a conserved distal side tyrosine

J Biol Chem. 2003 May 30;278(22):20185-91. doi: 10.1074/jbc.M211625200. Epub 2003 Mar 20.

Abstract

Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors. Exchange of tyrosine 249 in Synechocystis KatG, a distal side residue found in all as yet sequenced KatGs, had dramatic consequences on the bifunctional activity and the spectral features of the redox intermediate compound II. The Y249F variant lost catalase activity but retained a peroxidase activity (substrates o-dianisidine, pyrogallol, guaiacol, tyrosine, and ascorbate) similar to the wild-type protein. In contrast to wild-type KatG and similar to monofunctional peroxidases, the formation of the redox intermediate compound I could be followed spectroscopically even by addition of equimolar hydrogen peroxide to ferric Y249F. The corresponding bimolecular rate constant was determined to be (1.1 +/- 0.1) x 107 m-1 s-1 (pH 7 and 15 degrees C), which is typical for most peroxidases. Additionally, for the first time a clear transition of compound I to an oxoferryl-like compound II with peaks at 418, 530, and 558 nm was monitored when one-electron donors were added to compound I. Rate constants of reaction of compound I and compound II with tyrosine ((5.0 +/- 0.3) x 104 m-1 s-1 and (1.7 +/- 0.4) x 102 m-1 s-1) and ascorbate ((1.3 +/- 0.2) x 104 m-1 s-1 and (8.8 +/- 0.1) x 101 m-1 s-1 at pH 7 and 15 degrees C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins*
  • Base Sequence
  • Circular Dichroism
  • Cyanobacteria / enzymology*
  • DNA Primers
  • Electron Spin Resonance Spectroscopy
  • Kinetics
  • Molecular Sequence Data
  • Peroxidases / chemistry
  • Peroxidases / metabolism*
  • Sequence Homology, Amino Acid
  • Spectrophotometry, Ultraviolet
  • Tyrosine / metabolism*

Substances

  • Bacterial Proteins
  • DNA Primers
  • Tyrosine
  • Peroxidases
  • catalase HPI