The role of distal tryptophan in the bifunctional activity of catalase-peroxidases

Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelmin g catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate comp ound I by probing the role of the conserved tryptophan at the distal haem c avity site. Two wild-type proteins and three mutants of Synechocystis catal ase-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 m utations did not influence both formation of compound I and its reduction b y peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceede d that of the wild-type enzyme. This study demonstrates that the indole rin g of distal Trp in catalase-peroxidases is essential for the two-electron r eduction of compound I by hydrogen peroxide but not for compound I formatio n or for peroxidase reactivity (i.e. the one-electron reduction of compound I).