Low catalytic turnover of horseradish peroxidase in thiocyanate oxidation. Evidence for concurrent inactivation by cyanide generated through one-electron oxidation of thiocyanate.
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 alpha-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 (aN = 15.0 G and abetaH = 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.
Adak
S
S
Department of Physiology, Indian Institute of Chemical Biology, Calcutta 700 032, India.
Mazumdar
A
A
Banerjee
R K
RK
D001665:Binding Sites; D002384:Catalysis; D003486:Cyanides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D010084:Oxidation-Reduction; D019163:Reducing Agents / Q000494:pharmacology; D013053:Spectrophotometry; D013113:Spin Labels; D013861:Thiocyanates / Q000378:metabolism*
D003486:Cyanides; D004791:Enzyme Inhibitors; D005609:Free Radicals; D019163:Reducing Agents; D013113:Spin Labels; D013861:Thiocyanates; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; C031760:thiocyanate