EVIDENCE FOR A FREE-RADICAL CHAIN MECHANISM IN THE REACTION BETWEEN PEROXIDASE AND INDOLE-3-ACETIC-ACID AT NEUTRAL PH

The oxidation of indole-3-acetic acid (IAA) catalyzed by horseradish p eroxidase (HRP) in the absence of added H2O2 was studied at pH 7.4 usi ng spectral and kinetic approaches. Upon addition of a hundred-fold ex cess of IAA to HRP the native enzyme was rapidly transformed to compou nd II (HRP-II). HRP-II was the predominant catalytic enzyme species du ring the steady state. No compound III was observed. HRP-II was slowly transformed to the stable inactive verdohemoprotein, P-670. A precurs or of P-670, so-called P-940 was not detected. After the cessation of IAA oxidation there was neither oxygen consumption nor P-670 formation ; the remaining HRP-II was spontaneously reduced to native enzyme. Sin gle exponential kinetics were observed in the steady state for IAA oxi dation, oxygen consumption and P-670 formation yielding identical firs t order rate constants of about 6 . 10(4) s(-1). A comparison of the r ate of IAA oxidation by HRP-II in the steady state and in the transien t state indicated that more than 1/3 of the IAA was oxidized non-enzym atically during the steady state, confirming that a free radical chain reaction is involved in the peroxidase-catalyzed oxidation of IAA. IA A oxidation stopped before IAA was completely consumed, which cannot b e ascribed to enzyme inactivation because 30-50% of the enzyme was sti ll active after the end of the reaction. Instead, incomplete IAA oxida tion is explained in terms of termination of the free radical chain re action. Bimolecular rate constants of IAA oxidation by HRP-I and HRP-I I determined under transient state conditions were (2.2 +/- 0.1) . 10( 3) M(-1) s(-1) and (2.3 +/- 0.2) . 10(2) M(-1) s(-1).