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J. Biol. Chem., Vol. 280, Issue 11, 10395-10402, March 18, 2005

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Cytochrome b₆ Arginine 214 of Synechococcus sp. PCC 7002, a Key Residue for Quinone-reductase Site Function and Turnover of the Cytochrome bf Complex^*

Matthew E. Nelson, Giovanni Finazzi, Qing Jun Wang¶||, Kelly A. Middleton-Zarka**, John Whitmarsh¶, and Toivo Kallas

From the Department of Biology and Microbiology, University of Wisconsin-Oshkosh, Oshkosh, Wisconsin 54901, Physiologie Membranaire et Moléculaire du Chloroplaste, CNRS UPR 1261, Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France, and the ^¶Center of Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801

Quinone-reductase (Q_i) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q_i site mechanism remains largely enigmatic. To investigate the bf Q_i domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b₆ polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b₆ reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q_i site, mimicking the effect of the Q_i site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b₆ oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b₆ oxidation at the Q_i site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E_m,7) of the two b hemes similar to those in the wild type. Our data define cytochrome b₆ Arg²¹⁴ as a key residue for Q_i site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg²¹⁴ lies near the Q_i pocket and the newly discovered c_i or x heme. We propose a model for Q_i site function and a role for Arg²¹⁴ in plastoquinone binding.

Received for publication, September 23, 2004 , and in revised form, December 9, 2004.

^* This work was supported by National Science Foundation Grant MCB-0091415 and University of Wisconsin-Oshkosh Faculty Development Board Grant R788 (to T. K.) and by funds from the Centre National de la Recherche Scientifique (France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Mass Spectrometry Lab, Rockefeller University, 1230 York Ave., New York, NY 10021.

^** Present address: Potato Breeding and Genetics, Michigan State University, Crop and Soil Science Dept., East Lansing, MI 48824.

Present address: NIGMS, National Institutes of Health, Center for Bioinformatics and Computational Biology, 45 Center Drive, Bethesda, MD 20892-6200.

To whom correspondence should be addressed. Tel.: 920-424-7084; E-mail: kallas{at}uwosh.edu.

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