| pubmed-article:11724589 | pubmed:abstractText | The mechanism of the reaction of horseradish peroxidase isoenzyme C (HRPC) with hydrogen peroxide to form the reactive enzyme intermediate compound I has been studied using electronic absorbance, rapid-scan stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies at both acid and basic pH. The roles of the active site residues His42 and Arg38 in controlling heterolytic cleavage of the H(2)O(2) oxygen-oxygen bond have been probed with site-directed mutant enzymes His42 --> Leu (H42L), Arg38 --> Leu (R38L), and Arg38 --> Gly (R38G). The biphasic reaction kinetics of H42L with H(2)O(2) suggested the presence of an intermediate species and, at acid pH, a reversible second step, probably due to a neutral enzyme-H(2)O(2) complex and the ferric-peroxoanion-containing compound 0. EPR also indicated the formation of a protein radical situated more than approximately 10 A from the heme iron. The stoichiometry of the reaction of the H42L/H(2)O(2) reaction product and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) was concentration dependent and fell from a value of 2 to 1 above 0.7 mM ABTS. These data can be explained if H(2)O(2) undergoes homolytic cleavage in H42L. The apparent rate of compound I formation by H42L, while low, was pH independent in contrast to wild-type HRPC where the rate falls at acid pH, indicating the involvement of an ionizable group with pK(a) approximately 4. In R38L and R38G, the apparent pK(a) was shifted to approximately 8 but there is no evidence that homolytic cleavage of H(2)O(2) occurs. These data suggest that His42 acts initially as a proton acceptor (base catalyst) and then as a donor (acid catalyst) at neutral pH and predict the observed slower rate and lower efficiency of heterolytic cleavage observed at acid pH. Arg38 is influential in lowering the pK(a) of His42 and additionally in aligning H(2)O(2) in the active site, but it does not play a direct role in proton transfer. | lld:pubmed |
