EVIDENCE THAT CELLOBIOSE OXIDASE FROM PHANEROCHAETE-CHRYSOSPORIUM IS PRIMARILY AN FE(III) REDUCTASE - KINETIC COMPARISON WITH NEUTROPHIL NADPH OXIDASE AND YEAST FLAVOCYTOCHROME-B2
EVIDENCE THAT CELLOBIOSE OXIDASE FROM PHANEROCHAETE-CHRYSOSPORIUM IS PRIMARILY AN FE(III) REDUCTASE - KINETIC COMPARISON WITH NEUTROPHIL NADPH OXIDASE AND YEAST FLAVOCYTOCHROME-B2
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DOI：
10.1111/j.1432-1033.1992.tb16760.x
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发表时间：
1992-04-01
期刊：
影响因子：
--
通讯作者：
WOOD, PM
中科院分区：
文献类型：
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作者：
KREMER, SM;WOOD, PM
动力学测量进行了纯化的纤维二糖氧化酶在100 mM醋酸盐（pH 4.0）在30 ℃，过量的纤维二糖作为底物和O2或Fe（III）作为受体。 以230 μ M的O2作为唯一的电子受体，O2吸收速率对应于0.13 +/- 0.01 s-1的单电子转换数。 在不同O2浓度下的测量表明K（m）（O2）> 120 μ M。 在单独的实验中，在不存在氧气的情况下在340 nm处监测乙酸铁（III）的还原。 Fe（III）-乙酸盐还原的最大速度（V（max））为4.5 +/- 0.7 s-1，而K（m）[Fe（III）乙酸盐]为34 +/- 12 μ-M。 用铁氰化物代替Fe（III）乙酸盐，相应的值为6.9 +/- 0.7 s-1和23 +/- 5 μ M。 氧化还原滴定确定了在pH 4.0时氧化酶血红素辅基的电位为+165 mV。 Fe（III）/Fe（II）乙酸盐在pH 4.0时的中点电位要高得多，用200 μ M Fe得到的值为+535 mV。 纤维二糖氧化酶类似于酵母黄细胞色素b2，与中性粒细胞NADPH氧化酶的不同之处在于其血红素基团的电位远高于O2单电子还原为超氧化物的电位（E（m，4）= -110 mV）。 动力学比较得出的结论是，纤维二糖氧化酶的作用是作为一个铁（III）还原酶。 Fe（II）作为芬顿试剂[Fe（II）/H2 O2]的组分可能具有生物重要性。 收获时生长培养基中纤维二糖氧化酶的浓度（0.3 μ M）可以提供比文献中的非酶提议高得多的Fe（II）通量。
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.