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 microM 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 Km(O2) greater than 120 microM. 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 (Vmax) was 4.5 +/- 0.7 s-1, while Km[Fe(III) acetate] was 34 +/- 12 microM. With ferricyanide in place of Fe(III) acetate, the corresponding values were 6.9 +/- 0.7 s-1 and 23 +/- 5 microM. 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 microM 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 (Em,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 microM) can provide a far higher flux of Fe(II) than a non-enzymic proposal in the literature.
Kremer
S M
SM
Department of Biochemistry, University of Bristol, England.
Wood
P M
PM
D001487:Basidiomycota / Q000201:enzymology*; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D002475:Cellobiose / Q000378:metabolism; D038181:FMN Reductase; D006418:Heme / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D002475:Cellobiose; D006418:Heme; D007501:Iron; D002237:Carbohydrate Dehydrogenases; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); C019858:cellobiose oxidase; D038181:FMN Reductase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C023903:ferric citrate iron reductase; D010100:Oxygen