The pre-steady-state kinetics of inter-domain electron transfer in the extracellular flavocytochrome cellobiose dehydrogenase from Phanerochaete chrysosporium was studied using various values of pH and substrate concentration. Monitoring at the isosbestic point of each prosthetic group indicated that the reductive half-reactions of flavin and haem were biphasic and monophasic respectively. When the observed rates of the flavin and haem reactions were plotted against substrate concentration, the behaviour of the second phase of the flavin reduction was almost identical with that of haem reduction at all substrate concentrations and pH values tested, suggesting that the formation of flavin semiquinone and haem reduction involve the same electron transfer reaction. Although flavin reduction by cellobiose was observed in the range of pH3.0–7.0, the velocity of the next electron transfer step decreased with increase of pH and was almost zero above pH6.0. The second phase of flavin reduction and the haem reduction were inhibited similarly by high concentrations of the substrate, whereas the first phase of flavin reduction showed a hyperbolic relation to the cellobiose concentration. Increase in pH enhanced the substrate inhibition of haem reduction but not the initial flavin reduction. Moreover, the dissociation constant Kd of flavin reduction and the substrate inhibition constant Ki of haem reduction decreased similarly with an increase of pH. From these results, it is evident that binding of cellobiose to the active site inhibits electron transfer from flavin to haem.
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July 2002
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Research Article|
July 15 2002
Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium
Kiyohiko IGARASHI;
Kiyohiko IGARASHI
∗Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
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Ikuo MOMOHARA;
Ikuo MOMOHARA
†Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan
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Takeshi NISHINO;
Takeshi NISHINO
‡Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan
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Masahiro SAMEJIMA
Masahiro SAMEJIMA
1
∗Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
1To whom correspondence should be addressed (e-mail amsam@mail.ecc.u-tokyo.ac.jp).
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Publisher: Portland Press Ltd
Received:
December 10 2001
Revision Received:
March 18 2002
Accepted:
April 08 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 365 (2): 521–526.
Article history
Received:
December 10 2001
Revision Received:
March 18 2002
Accepted:
April 08 2002
Citation
Kiyohiko IGARASHI, Ikuo MOMOHARA, Takeshi NISHINO, Masahiro SAMEJIMA; Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Biochem J 15 July 2002; 365 (2): 521–526. doi: https://doi.org/10.1042/bj20011809
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