EVIDENCE THAT CELLOBIOSE OXIDASE FROM PHANEROCHAETE-CHRYSOSPORIUM IS PRIMARILY AN FE(III) REDUCTASE - KINETIC COMPARISON WITH NEUTROPHIL NADPH OXIDASE AND YEAST FLAVOCYTOCHROME-B2

Kinetic measurements were made for purified cellobiose oxidase in 100 mM acetate (pH 4.0) at 30-degrees-C, with excess cellobiose as substrate and O2 or Fe(III) as acceptor. With O2 at 230-mu-M 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-mu-M. 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-mu-M. With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 +/- 0.7 s-1 and 23 +/- 5-mu-M. Redox titrations established the potential of the haem 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-mu-M 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-mu-M) can provide a far higher flux of Fe(II) than a non-enzymic proposal in the literature.