The role of distal tryptophan in the bifunctional activity of catalase-peroxidases.
Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).
Regelsberger
G
G
Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Jakopitsch
C
C
Furtmüller
P G
PG
Rueker
F
F
Switala
J
J
Loewen
P C
PC
Obinger
C
C
D001426:Bacterial Proteins; D001665:Binding Sites; D002384:Catalysis; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D006418:Heme; D014364:Tryptophan; D006861:Hydrogen Peroxide; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria; D003578:Cytochrome-c Peroxidase; C076136:catalase HPI