The inactivation and catalytic pathways of horseradish peroxidase with m-chloroperoxybenzoic acid. A spectrophotometric and transient kinetic study.
The kinetics of the catalytic cycle and irreversible inactivation of horseradish peroxidase C (HRP-C) reacting with m-chloroperoxybenzoic acid (mCPBA) have been studied by conventional and stopped-flow spectrophotometry. mCPBA oxidized HRP-C to compound I with a second order-rate constant k1 = 3.6 x 10(7) M-1 s-1 at pH 7.0, 25 degrees C. Excess mCPBA subsequently acted as a one-electron reducing substrate, converting compound I to compound II and compound II to resting, ferric enzyme. In both of these reactions, spectrally distinct, transient forms of the enzyme were observed (lambdamax = 411 nm, epsilon = 45 mM-1 cm-1 for compound I with mCPBA, and lambdamax = 408 nm, epsilon = 77 mM-1 cm-1 for compound II with mCPBA). The compound I-mCPBA intermediate (shown by near infrared spectroscopy to be identical to P965) decayed either to compound II in a catalytic cycle (k3 = 6.4 x 10(-3) s-1) or, in a competing inactivation reaction, to verdohemoprotein (ki = 3.3 x 10(-3) s-1). Thus, a partition ratio of r = 2 is obtained for the inactivation of ferric HRP-C by mCPBA. The intermediate formed from compound II with mCPBA is not part of the inactivation pathway and only decays via the catalytic cycle to give resting, ferric enzyme (k5 = 1.0 x 10(-3) s-1). The data are compared with those from earlier steady-state kinetic studies and demonstrate the importance of single turnover experiments. The results are discussed in terms of the physiologically relevant reactions of plant peroxidases with hydrogen peroxide.
Rodriguez-Lopez
J N
JN
Nitrogen Fixation Laboratory, John Innes Centre, NR4 7UH Norwich, United Kingdom.
Hernández-Ruiz
J
J
Garcia-Cánovas
F
F
Thorneley
R N
RN
Acosta
M
M
Arnao
M B
MB
D002021:Buffers; D002723:Chlorobenzoates / Q000494:pharmacology*; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007700:Kinetics; D010710:Phosphates; D013054:Spectrophotometry, Atomic; D013056:Spectrophotometry, Ultraviolet
D002021:Buffers; D002723:Chlorobenzoates; D007202:Indicators and Reagents; D007527:Isoenzymes; D010710:Phosphates; D006735:Horseradish Peroxidase; C000433:3-chloroperbenzoic acid; C018279:sodium phosphate