Low catalytic turnover of horseradish peroxidase in thiocyanate oxidation

Abstract

The catalytic turnover of horseradish peroxidase (HRP) to oxidize SCN^- is a hundredfold lower than that of lactoperoxidase (LPO) at optimum pH. While studying the mechanism, HRP was found to be reversibly inactivated following pseudo-first order kinetics with a second order rate constant of 400 M^-1 min^-1 when incubated with SCN^- and H₂O₂. The slow rate of SCN^- oxidation is increased severalfold in the presence of free radical traps, 5-5-dimethyl-1-pyrroline N-oxide or α-phenyl-tert-butylnitrone, suggesting the plausible role of free radical or radical-derived product in the inactivation. Spectral studies indicate that SCN^- at a lower concentrations slowly reduces compound II to native state by one-electron transfer as evidenced by a time-dependent spectral shift from 418 to 402 nm through an isosbestic point at 408 nm. In the presence of higher concentrations of SCN^-, a new stable Soret peak appears at 421 nm with a visible peak at 540 nm, which are the characteristics of the inactivated enzyme. The one-electron oxidation product of SCN^- was identified by electron spin resonance spectroscopy as 5-5-dimethyl-1-pyrroline N-oxide adduct of the sulfur-centered thiocyanate radical (a^N = 15.0 G and aβ^H = 16.5 G). The inactivation of the enzyme in the presence of SCN^- and H₂O₂ is prevented by electron donors such as iodide or guaiacol. Binding studies indicate that both iodide and guaiacol compete with SCN^- for binding at or near the SCN^- binding site and thus prevent inactivation. The spectral characteristics of the inactivated enzyme are exactly similar to those of the native HRP-CN^- complex. Quantitative measurements indicate that HRP produces a 10-fold higher amount of CN^- than LPO when incubated with SCN^- and H₂O₂. As HRP has higher affinity for CN^- than LPO, it is concurrently inactivated by CN^- formed during SCN^- oxidation, which is not observed in case of LPO. This study further reveals that HRP catalyzes SCN^- oxidation by two one-electron transfers with the intermediate formation of thiocyanate radicals. The radicals dimerize to form thiocyanogen, (SCN)₂, which is hydrolyzed to form CN^-. As LPO forms OSCN^- as the major stable oxidation product through a two-electron transfer mechanism, it is not significantly inactivated by CN^- formed in a small quantity.