Abstract
Catalase-peroxidases have a predominant catalase activity but differ from monofunctional catalases in exhibiting a substantial peroxidase activity and in having different residues in the heme cavity. We present a kinetic study of the formation of the key intermediate compound I by probing the role of the conserved distal amino acid triad Arg-Trp-His of a recombinant catalase-peroxidase in its reaction with hydrogen peroxide, peroxoacetic acid, and m-chloroperbenzoic acid. Both the wild-type enzyme and six mutants (R119A, R119N, W122F, W122A, H123Q, H123E) have been investigated by steady-state and stopped-flow spectroscopy. The turnover number of catalase activity of R119A is 14.6%, R119N 0.5%, H123E 0.03%, and H123Q 0.02% of wild-type activity. Interestingly, W122F and W122A completely lost their catalase activity but retained their peroxidase activity. Bimolecular rate constants of compound I formation of the wild-type enzyme and the mutants have been determined. 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 spectroscopically underlining the important role of Trp-122 in catalase activity. The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide (i.e. compound I formation), whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide.
Highlights
Catalase-peroxidases (KatGs)1 cover a growing group of enzyme
The results demonstrate that the role of the distal His-Arg pair in catalase-peroxidases is important in the heterolytic cleavage of hydrogen peroxide, whereas the distal tryptophan is essential for compound I reduction by hydrogen peroxide
We have shown that the spectrum of KatG compound I is reminiscent to ascorbate peroxidases (APX) compound I [7] with the important difference that compound I formation of KatG could never be monitored with hydrogen peroxide
Summary
Catalase-peroxidases (KatGs) cover a growing group of enzyme. They are components of the oxidative defense system of bacterial [1] and fungal [2, 3] cells and function primarily as catalases to remove hydrogen peroxide before it can damage cellular components. KatGs function as broad specificity peroxidases, oxidizing various electron donors, including NAD(P)H (8 –10), phenols, and anilines [7], whereas typical substrates for APX and CCP (ascorbate and cytochrome c, respectively) are extremely poor electron donors for KatGs [5,6,7, 11] From both CCP and APX, the three-dimensional structures are known [12, 13] and exhibit highly conserved amino acid residues at the active site. Mutation of distal arginine and histidine gave 10- to 102-fold and 104-fold decreases in catalase activity, indicating a role in O-O heterolysis of hydrogen peroxide Their role in compound I formation with peroxy acids was much less pronounced
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