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Journal of Biochemistry Advance Access originally published online on January 18, 2007
Journal of Biochemistry 2007 141(3):377-387; doi:10.1093/jb/mvm042
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© 2007 The Japanese Biochemical Society.

Inhibition of Reversed Electron Transfer and Proton Transport in the Beef Heart Cytochrome bc1 Complex by Chemical Modification

Toshiaki Miki1,*, Masato Umeda2 and Yoshie Harada1,3

1The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan; 2Division of Molecular Biology and Information, Institute for Chemical Research, Kyoto University, Kyoto 611-0011, Japan and 3Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Honcho, Kawaguchi, Saitama 332-0012, Japan

*To whom correspondence should be addressed. Tel: +81-3-3823-2105, Fax: +81-3-3823-2965, E-mail: miki{at}rinshoken.or.jp

Received September 20, 2006; Accepted January 11, 2007


  Abstract

Chemical modification of the bovine heart cytochrome bc1 complex with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) has been reported to inhibit the proton pumping activity without affecting the rate of electron transfer to ferricytochrome c. This study aims to examine the effect of EEDQ on energy-linked reversed electron transfer in the bc1 complex reconstituted into potassium-loaded phospholipid vesicles. Generation of a valinomycin-mediated potassium-diffusion potential induced the reduction of cytochrome b in the reconstituted bc1 complex in the presence of sodium ascorbate. The time course of the cytochrome b reduction was well correlated with that of the absorbance change of safranine, an optical probe for measuring membrane potential. Treatment of the bc1 complex with EEDQ caused a decrease in the potential-induced reduction of cytochrome b as well as in the proton translocation activity. But a significant loss in the ubiquinol–cytochrome c reducing activity was not observed in the EEDQ-treated bc1 complex. The time- and concentration-dependent effect of EEDQ on the reversed electron transfer was well correlated with that of the proton translocation activity of the bc1 complex. These findings strongly support the idea that the potential-induced reversal of electron transfer is coupled to the reverse flow of protons in the cytochrome bc1 complex.

Key Words: chemical modification, cytochrome bc1 complex, proton transport, respiratory chain, reversed electron transfer

Abbreviations: DCCD, N,N '-dicyclohexylcarbodiimide; EEDQ, N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydro-quinoline; FCCP, carbonyl cyanide p-trifluoromethoxy phenylhydrazone; [2Fe–2S], iron–sulphur cluster; ISP, Rieske iron–sulphur protein; MOPS, 4-morpholinepropanesulfonic acid; PMS, phenazine methosulphate; Q2 and Q2H2, the oxidized and reduced form of ubiquinone-2; SMP, submitochondrial particles; TMPD, N,N,N ',N '-tetramethyl-p-phenylenediamine


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