Effect of distal cavity mutations on the formation of compound I in catalase-peroxidases

Catalase-peroxidases have a predominant catalase activity but differ from m onofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic stud y of the formation of the key intermediate compound I by probing the role o f the conserved distal amino acid triad Arg-Trp-Ris of a recombinant catala se-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, an d m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A , R119N, TY122F, W122A, H123Q, H123E) have been investigated by steady-stat e and stopped-flow spectroscopy. The turnover number of catalase activity o f R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type act ivity. Interestingly, W122F and W122A completely lost their catalase activi ty but retained their peroxidase activity. Bimolecular rate constants of co mpound I formation of the wild-type enzyme and the mutants have been determ ined. The Trp-122 mutants for the first time made it possible to follow the transition of the ferric enzyme to compound I by hydrogen peroxide spectro scopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalas e-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential fo r compound I reduction by hydrogen peroxide.