Abstract
With the exception of catalase-peroxidases, heme peroxidases show no significant ability to oxidize hydrogen peroxide and are trapped and inactivated in the compound III form by H2O2 in the absence of one-electron donors. Interestingly, some KatG variants, which lost the catalatic activity, form compound III easily. Here, we compared the kinetics of interconversion of ferrous enzymes, compound II and compound III of wild-type Synechocystis KatG, the variant Y249F, and horseradish peroxidase (HRP). It is shown that dioxygen binding to ferrous KatG and Y249F is reversible and monophasic with apparent bimolecular rate constants of (1.2 +/- 0.3) x 10(5) M(-1) s(-1) and (1.6 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C), similar to HRP. The dissociation constants (KD) of the ferrous-dioxygen were calculated to be 84 microm (wild-type KatG) and 129 microm (Y249F), higher than that in HRP (1.9 microm). Ferrous Y249F and HRP can also heterolytically cleave hydrogen peroxide, forming water and an oxoferryl-type compound II at similar rates ((2.4 +/- 0.3) x 10(5) M(-1) s(-1) and (1.1 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C)). Significant differences were observed in the H2O2-mediated conversion of compound II to compound III as well as in the spectral features of compound II. When compared with HRP and other heme peroxidases, in Y249F, this reaction is significantly faster ((1.2 +/- 0.2) x 10(4) M(-1) s(-1))). Ferrous wild-type KatG was also rapidly converted by hydrogen peroxide in a two-phasic reaction via compound II to compound III (approximately 2.0 x 10(5) M(-1) s(-1)), the latter being also efficiently transformed to ferric KatG. These findings are discussed with respect to a proposed mechanism for the catalatic activity.
Highlights
With the exception of catalase-peroxidases, heme peroxidases show no significant ability to oxidize hydrogen peroxide and are trapped and inactivated in the compound III form by H2O2 in the absence of one-electron donors
We compared the kinetics of interconversion of ferrous enzymes, compound II and compound III of wild-type Synechocystis KatG, the variant Y249F, and horseradish peroxidase (HRP)
Compound I does not accumulate during H2O2 degradation but can be trapped by using organic peroxides (8). This intermediate exhibits only a low reactivity toward H2O2 (8). Another peculiarity of KatGs regards the spectral features of compound II formed by one-electron reduction of compound I, which in most heme peroxidases is a ferryl species with a red-shifted Soret band and two typical maxima in the visible region (7) and accumulates in the peroxidase cycle
Summary
Neither its reactivity toward dioxygen (see Fig. 1, Reaction 6) nor its two-electron oxidation reaction to compound II mediated by H2O2 (see Fig. 1, Reaction 10) (12) nor the transition of compound II to compound III has been investigated so far, the knowledge of these reactions could help to understand the KatG-specific high catalase activity This is most obvious when looking at the. The catalase activity was dramatically decreased, the formation of (conventional) compound I could be followed even with equimolar H2O2, and a typical (ferryl-)compound II was formed, which was transformed to compound III (13, 14) These findings motivated us to perform the following comparative kinetic investigation of the reaction of ferrous wildtype KatG from Synechocystis, the variant Y249F, and horseradish peroxidase, respectively, with both dioxygen and hydrogen peroxide as well as of the interconversion of compound II to compound III. By using the stopped-flow technique, we were able to calculate the apparent bimolecular rate constants of the transitions ferrous protein 3 compound III, ferrous protein 3 compound II, and compound II 3 compound III and detect significant differences between the three investigated protein species
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
