Total Conversion of Bifunctional Catalase-Peroxidase (KatG) to Monofunctional Peroxidase by Exchange of a Conserved Distal Side Tyrosine

Christa Jakopitsch, Markus Auer, Anabella Ivancich, Florian Rüker, Paul Georg Furtmüller, Christian Obinger
2003 Journal of Biological Chemistry  
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,
more » ... 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) ؋ 10 7 M ؊1 s ؊1 (pH 7 and 15°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) ؋ 10 4 M ؊1 s ؊1 and (1.7 ؎ 0.4) ؋ 10 2 M ؊1 s ؊1 ) and ascorbate ((1.3 ؎ 0.2) ؋ 10 4 M ؊1 s ؊1 and (8.8 ؎ 0.1) ؋ 10 1 M ؊1 s ؊1 at pH 7 and 15°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. Catalase-peroxidases (KatGs) 1 are found in archaebacteria, eubacteria, and fungi. On the basis of sequence similarities with fungal cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs), KatGs have been shown to be members of class I of the superfamily of plant, fungal, and bacterial heme peroxidases (1). Recently, the 2.0-Å crystal structure of the KatG from the archaebacterium Haloarcula marismortui has
doi:10.1074/jbc.m211625200 pmid:12649295 fatcat:nz6wpflqtjay5ocvryjcjlsqly