Tài liệu Báo cáo khoa học: Electron transfer chain reaction of the extracellular flavocytochrome

Electron transfer chain reaction of the extracellular

flavocytochrome cellobiose dehydrogenase from the

basidiomycete Phanerochaete chrysosporium

Kiyohiko Igarashi1

, Makoto Yoshida1

, Hirotoshi Matsumura2

, Nobuhumi Nakamura2

,

Hiroyuki Ohno2

, Masahiro Samejima1 and Takeshi Nishino3

1 Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan

2 Department of Biotechnology, Tokyo University of Agricultural and Technology, Japan

3 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan

Cellulose is the most abundant natural polymer on

earth, and its degradation is thus an important compo￾nent of the carbon cycle. Although cellulose is often

referred to as a b-linked glucose polymer, cellobiose, a

b-1,4-linked glucose dimer, should strictly be regarded

as the repeating unit of cellulose, because adjacent

glucoses show opposing faces to each other in the cel￾lulose chain [1,2]. Many microorganisms recognize this

repeating unit and hydrolyze cellulose to cellobiose as

an initial step in the metabolism [3]. In filamentous

fungi, cellulose degradation had been thought to pro￾ceed via two-step hydrolysis, i.e. cellulose is hydrolyzed

to cellobiose by various cellulases and the product is

further hydrolyzed to glucose by b-glucosidase. How￾ever, recent cytochemical, kinetic, and transcriptional

studies [4–6] have supported another hypothesis

Keywords

cellobiose dehydrogenase; cellulose

degradation; electron-transfer;

Phanerochaete chrysosporium

Correspondence

K. Igarashi, Department of Biomaterials

Sciences, Graduate School of Agricultural

and Life Sciences, The University of Tokyo,

Bunkyo-ku, Tokyo 113-8657, Japan

Fax: +81 3 5841 5273

Tel: +81 3 5841 5258

E-mail: [email protected]

(Received 27 February 2005, revised

26 March 2005, accepted 6 April 2005)

doi:10.1111/j.1742-4658.2005.04707.x

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome con￾taining flavin and b-type heme, and plays a key role in cellulose degrada￾tion by filamentous fungi. To investigate intermolecular electron transfer

from CDH to cytochrome c, Phe166, which is located in the cytochrome

domain and approaches one of propionates of heme, was mutated to Tyr,

and the thermodynamic and kinetic properties of the mutant (F166Y) were

compared with those of the wild-type (WT) enzyme. The mid-point

potential of heme in F166Y was measured by cyclic voltammetry, and was

estimated to be 25 mV lower than that of WT at pH 4.0. Although pre￾steady-state reduction of flavin was not affected by the mutation, the rate

of subsequent electron transfer from flavin to heme was halved in F166Y.

When WT or F166Y was reduced with cellobiose and then mixed with

cytochrome c, heme re-oxidation and cytochrome c reduction occurred syn￾chronously, suggesting that the initial electron is transferred from reduced

heme to cytochrome c. Moreover, in both enzymes the observed rate of

the initial phase of cytochrome c reduction was concentration dependent,

whereas the second phase of cytochrome c reduction was dependent on the

rate of electron transfer from flavin to heme, but not on the cytochrome c

concentration. In addition, the electron transfer rate from flavin to heme

was identical to the steady-state reduction rate of cytochrome c in both

WT and F166Y. These results clearly indicate that the first and second

electrons of two-electron-reduced CDH are both transferred via heme, and

that the redox reaction of CDH involves an electron-transfer chain mech￾anism in cytochrome c reduction.

Abbreviations

CDH, cellobiose dehydrogenase; F166Y, Phe166Tyr mutant CDH; NHE, normal hydrogen electrode; WT, wild-type CDH.

FEBS Journal 272 (2005) 2869–2877 ª 2005 FEBS 2869