Evidence that cellobiose oxidase from Phanerochaete chrysosporium is primarily an Fe(III) reductas

Abstract

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30°C, with excess cellobiose as substrate and O₂ or Fe(III) as acceptor. With O₂ at 230 μ as sole electron acceptor, the O₂ uptake rate corresponded to a one-electron turnover number of 0.13 ± 0.01 s⁻¹. Measurements at different O₂ concentrations indicated K_m(O₂) > 120 μM. In separate experiments, the reduction of Fe(III) acetate was monitored at 340 nm in the absence of oxygen. The maximum velocity of Fe(III)-acetate reduction (V_max) was 4.5 ± 0.7 s⁻¹, while K_m[Fe(III) acetate] was 34 ± 12 μM. With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 ± 0.7 s⁻¹ and 23 ± 5 μM. Redox titrations established the potential of the heam prosthetic group of the oxidase at pH 4.0 as + 165 mV. The midpoint potential for Fe(III)/Fe(II) acetate at pH 4.0 is much higher, a value of + 535 mV being obtained with 200 μM Fe. Cellobiose oxidase resembles yeast flavocytochrome b₂ and differs from the neutrophil NADPH oxidase in having the potential of its haem group far above the potential for one-electron reduction of O₂ to superoxide (E_m,4=−110 mV). A kinetic comparison led to the conclusion that the role of cellobiose oxidase is as an Fe(III) reductase. Fe(II) may have a biological importance as a component of Fenton's reagent [Fe(II)/H₂O₂]. The concentration of cellobiose oxidase in the growth medium at harvest (0.3 μM) can provide a far higher flux of Fe(II) than a non-enzymic proposal in the literature.