Experimental Evidence for Proton Motive Force-Dependent Catalysis by the Diheme-Containing Succinate:Menaquinone Oxidoreductase from the Gram-Positive Bacterium Bacillus licheniformis ² M. Gregor Madej, ‡ Hamid R. Nasiri, § Nicole S. Hilgendorff, ‡ Harald Schwalbe, § Gottfried Unden, | and C. Roy D. Lancaster* ,‡ Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-Von-Laue-Strasse 3, D-60438 Frankfurt am Main, Germany, Institut fu ¨r Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-UniVersita ¨t, Max-Von-Laue-Strasse 7, D-60438 Frankfurt am Main, Germany, and Institut fu ¨r Mikrobiologie und Weinforschung, Johannes Gutenberg-UniVersita ¨t Mainz, Becherweg 15, 55099 Mainz, Germany ReceiVed August 31, 2006; ReVised Manuscript ReceiVed October 5, 2006 ABSTRACT: In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Δp, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Δp dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme- containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/ reduction potentials, provide unequivocal evidence for Δp-dependent catalysis of succinate oxidation by quinone as well as for Δp generation upon catalysis of fumarate reduction by quinol. Succinate:quinone oxidoreductases (SQORs 1 )(1, 2) are integral membrane protein complexes, which couple the two- electron oxidation of succinate to fumarate (succinate f fumarate + 2H + + 2e - ; E M7 )+25 mV (3)) to the two- electron reduction of quinone to quinol (quinone + 2H + + 2e - f quinol) as well as catalyzing the opposite reaction, the reduction of fumarate by quinol (4). In mitochondria and some aerobic bacteria, succinate:ubiquinone reductase, also known as succinate dehydrogenase from the tricarboxylic acid (TCA or Krebs) cycle and as complex II of the aerobic respiratory chain, catalyzes the mildly exergonic reduction of succinate by ubiquinone (Q, E M7 (Q/QH 2 ) )+90 mV (3)), which is not directly associated with energy storage in the form of a transmembrane electrochemical proton potential (Δp). Gram-positive bacteria do not contain ubiquinone but rather the lower-potential menaquinone (MK (5), E M7 (MK/ MKH 2 ) )-74 mV (6)). In these cases, the catalyzed oxidation of succinate by quinone is endergonic under standard conditions (1, 7, 8). Consequently, these bacteria face a thermodynamic problem in supporting the catalysis of this reaction in ViVo. In Gram-positive bacteria, such as Bacillus subtilis and Bacillus licheniformis and other prokaryotes containing succinate:menaquinone reductases, the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Δp, is abolished by the rupture of the bacteria or by the addition of a protonophore (7, 8, 9). It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential (8). The protons ² This work was supported by the Deutsche Forschungsgemeinschaft (SFB 472 “Molecular Bioenergetics”) and the Max Planck Society. * To whom correspondence should be addressed. Tel: +49-69-6303- 1013. Fax: +49-69-6303-1002. E-mail: Roy.Lancaster@mpibp-frank- furt.mpg.de. ‡ Max Planck Institute of Biophysics. § Johann Wolfgang Goethe-Universita ¨t. | Johannes Gutenberg-Universita ¨t Mainz. 1 Abbreviations: C12E9, polyoxyethylene 9-dodecyl ether; CCCP, carbonyl cyanide m-chloro-phenylhydrazone; DDM: n-dodecyl--D- maltoside; DMN, 2,3-dimethyl-1,4-naphthoquinone; DMNH2, 2,3- dimethyl-1,4-naphthoquinol; DTE, dithioerythritol; DTT, dithiothreitol; EMN, 2-ethyl-3-methyl-1,4-naphthoquinone; EMNH2, 2-ethyl-3-methyl- 1,4-naphthoquinol; EQ-0, 2,3-dimethoxy-5-ethyl-6-methyl-1,4-benzo- quinone; EQH2-0, 2,3-dimethoxy-5-ethyl-6-methyl-1,4-benzoquinol; FAD, flavin adenine dinucleotide; Fum, fumarate; HNN, 2-hydroxy- 3-neopentyl-1,4-naphthoquinone; MMAN, 2-methyl-3-methylamino- 1,4-naphthoquinone; MMANH2, 2-methyl-3-methylamino-1,4-naph- thoquinol; MOPS, 3-[N-morpholino] propanesulfonic acid; PL, phospholipid; QFR, quinol:fumarate reductase; SQR, succinate:quinone reductase; SQOR, succinate:quinone oxidoreductase. 15049 Biochemistry 2006, 45, 15049-15055 10.1021/bi0618161 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/23/2006