Pseudoperoxidase activity of myoglobin:: Kinetics and mechanism of the peroxidase cycle of myoglobin with H2O2 and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) as substrates

Using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate, it has been shown that the increased peroxidase activity for decreasing pH of myoglobin activated by hydrogen peroxide is due to a protonization of ferrylmyoglobin, MbFe(IV)=O, facilitating electron transfer from the substrate and corresponding to pK(a) similar to 5.2 at 25.0 degreesC and ionic strength 0.16, rather than due to specific acid catalysis. On the basis of stopped flow absorption spectroscopy with detection of the radical cation ABTS(.+), the second-order rate constant and activation parameters for the reaction between MbFe(IV)=O and ABTS were found to have the values k = 698 +/- 32 M-1 s(-1), DeltaH(#) = 66 +/- 4 kJ mol(-1), and DeltaS(#) = 30 +/- 15 J mol(-1) K-1 at 25.0 degreesC and physiological pH (7.4) and ionic strength (= 0.16 M NaCl). At a lower pH (5.8) corresponding to the conditions in meat, values were found as follows: k = 3.5 +/- 0.3 x 10(4) M-1 s(-1), DeltaH(#) = 31 +/- 6 kJ mol(-1), and DeltaS(#) = -53 +/- 19 J mol(-1) K-1, indicative of a shift from outersphere electron transfer to an innersphere mechanism. For steady state assay conditions, this shift is paralleled by a shift from saturation kinetics at pH 7.4 to first-order kinetics for H2O2 as substrate at pH 5.8. In contrast, the activation reaction between myoglobin and hydrogen peroxide was found at 25.0 degreesC to be slow and independent of pH with values of 171 +/- 7 and 196 +/- 19 M-1 s(-1) found at physiological and meat pH, respectively, as determined by sequential stopped flow spectroscopy, from which a lower limit of k = 6 x 10(5) M-1 s(-1) for the reaction between perferrylmyoglobin, .MbFe(IV)=O, and ABTS could be estimated. As compared to the traditional peroxidase assay, a better characterization of pseudoperoxidase activity of heme pigments and their denatured or proteolyzed forms is thus becoming possible, and specific kinetic effects on activation, substrate oxidation, or shift in rate determining steps may be detected.