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Volume 272, Number 17, Issue of April 25, 1997 pp. 11049-11056
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Low Catalytic Turnover of Horseradish Peroxidase in Thiocyanate Oxidation
EVIDENCE FOR CONCURRENT INACTIVATION BY CYANIDE GENERATED THROUGH ONE-ELECTRON OXIDATION OF THIOCYANATE

(Received for publication, August 17, 1996, and in revised form, January 19, 1997)

From the Department of Physiology, Indian Institute of Chemical Biology, Calcutta 700 032, India

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 M¹ min¹ 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 -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 a^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.

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