SPECTRAL AND KINETIC-PROPERTIES OF OXIDIZED INTERMEDIATES OF COPRINUS-CINEREUS PEROXIDASE

Spectra of native Coprinus cinereus peroxidase and its oxidized intermediates, compounds I, II and III, the kinetics of compound I formation and guaiacol activity of the enzyme have been studied at 25-degrees-C. Mechanistic aspects of the oxidation of the enzyme and its oxidized intermediates indicate that the enzyme operates via the classic peroxidatic cycle. Isosbestic points in the UV-VIS range between native enzyme-compound I are 353, 426, 453 and 547 nm, between native enzyme-compound II 343, 414, 465, 530, 629 and 668 nm, between native enzyme-compound III 344, 411, 475, 534 and 604 nm and compound I-compound II 394 and 576 nm. Soret-region maximum molar absorption coefficients [mM-1 cm-1] with wavelengths of maximum absorption in brackets are for the native enzyme 109 (405 nm), for compound I 63 (402 nm), for compound II 92 (419 nm) and for compound III 105 (417 nm). The native enzyme exhibited a shift in the Soret absorption maximum wavelength from 393 to 405 nm over the pH range 3.58-6.01. No shift occurred from pH 6.01-10.83. The pH dependence of compound I formation showed the presence of an acidic heme-linked group in the enzyme with a pK(a) of 4.9 +/- 0.1. Hydrogen peroxide reacts with the basic form of the native enzyme, forming compound I with a second-order rate constant of (7.1 +/- 0.1) x 10(6) M-1 s-1. Guaiacol activity of the enzyme reaches a maximum at pH 8.