Differences in Protonation of Ubiquinone and Menaquinone in Fumarate Reductase from Escherichia coli

Abstract

Escherichia coli quinol-fumarate reductase operates with both natural quinones, ubiquinone (UQ) and menaquinone (MQ), at a single quinone binding site. We have utilized a combination of mutagenesis, kinetic, EPR, and Fourier transform infrared methods to study the role of two residues, Lys-B228 and Glu-C29, at the quinol-fumarate reductase quinone binding site in reactions with MQ and UQ. The data demonstrate that Lys-B228 provides a strong hydrogen bond to MQ and is essential for reactions with both quinone types. Substitution of Glu-C29 with Leu and Phe caused a dramatic decrease in enzymatic reactions with MQ in agreement with previous studies, however, the succinate-UQ reductase reaction remains unaffected. Elimination of a negative charge in Glu-C29 mutant enzymes resulted in significantly increased stabilization of both UQ . and MQ . semiquinones. The data presented here suggest similar hydrogen bonding of the C1 carbonyl of both MQ and UQ, whereas there is different hydrogen bonding for their C4 carbonyls. The differences are shown by a single point mutation of Glu-C29, which transforms the enzyme from one that is predominantly a menaquinol-fumarate reductase to one that is essentially only functional as a succinate-ubiquinone reductase. These findings represent an example of how enzymes that are designed to accommodate either UQ or MQ at a single Q binding site may nevertheless develop sufficient plasticity at the binding pocket to react differently with MQ and UQ.Facultative anaerobic bacteria and lower eukaryotes adapt their metabolism in response to environmental changes. An important aspect of this adaptability is that many can synthesize both ubiquinone (UQ) 4 and menaquinone (MQ). Quinone biosynthesis and relative concentration is regulated by growth conditions and oxygen supply with UQ and MQ being the primary quinone under aerobic and anaerobic conditions, respectively (1). Membrane-bound bacterial enzymes that utilize quinones as substrates can often catalyze redox reactions with both UQ and MQ. Membrane-bound quinol-fumarate reductase (QFR) in Escherichia coli is an example of an enzyme that can readily use both types of quinones and shows high menaquinolfumarate and succinate-quinone reductase activities (2). QFR serves as a terminal reductase in the anaerobic bacterial respiratory chain by catalyzing the menaquinol-fumarate reductase reaction (3). When genetically manipulated to allow its expression under aerobic conditions, QFR efficiently replaces succinateubiquinone reductase (SQR) in aerobic metabolism and cell growth by catalyzing ubiquinone reduction from succinate (4).Membrane-bound QFR from E. coli is a four subunit complex, and its x-ray structure has been solved (5-9). The FrdA and FrdB subunits comprise the soluble component that contains a dicarboxylate substrate binding site, a covalently bound FAD, and three linearly arranged iron-sulfur centers (5-7). The membrane-spanning hydrophobic subunits FrdC and FrdD are necessary to anchor the soluble the FrdAB domain to the membrane an...

Plasticity of the Quinone-binding Site of the Complex II Homolog Quinol:Fumarate Reductase

“…Kinetic and spectroscopic investigations of these two amino acids showed that Lys-B228 was essential for both menaquinol-fumarate reductase activity as well as succinate-ubiquinone reductase activity. Eliminating the negative charge of Glu-C29 also resulted in increased stabilization of both UQ ⅐Ϫ and MQ ⅐Ϫ semiquinones (16,19). Interestingly, mutation of Glu-C29, although having a profound effect on menaquinol-fumarate reductase activity, had only negligible effects on succinate-ubiquinone reductase activity (16).…”

“…Interestingly, biochemical studies highlight differences in potential pathways by which MQ and UQ are deprotonated and protonated, respectively, in QFR (16). This suggests that there may be differences in amino acid residues that participate in the different activities with quinones.…”

“…In QFR, the quinone-binding domain comprises the membrane-bound subunits FrdC (ϳ15 kDa) and FrdD (ϳ13 kDa) as well as amino acid residues from the iron-sulfur subunit FrdB. Structural and biochemical analyses have shown that QFR can accommodate both MQ and UQ at a single quinone-catalytic site termed Q P (12,13,15,16).…”

“…This suggests that there may be differences in amino acid residues that participate in the different activities with quinones. Two amino acid residues were shown to be essential for proficient menaquinol-fumarate reductase activity (12,16,17). Lys-B228, where the "B" indicates that this residue is part of the FrdB subunit, was shown to form a strong hydrogen bond to the C1 carbonyl oxygen of MQ, and Glu-C29 forms a hydrogen bond to the C4 carbonyl oxygen of MQ (12,16,18) (Fig.…”

Menaquinone as Well as Ubiquinone as a Crucial Component in the Escherichia coli Respiratory Chain

“…The former is involved in oxidation reaction of succinate coupled with the reduction of UQ under aerobic conditions, and the latter functions in reducing fumarate and oxidizing reduced MK under anaerobic conditions. The cells might perform an efficient reaction by a combination of these enzymes and types of isoprenoid quinones, which are differently synthesized or produced under the two different conditions (Maklashina et al, 2006).…”