Factors governing the efficacy of long-range electron relays in enzymes have been examined using protein film voltammetry in conjunction with site-directed mutagenesis. Investigations of the fumarate reductase from Escherichia coli, in which three Fe-S clusters relay electrons over more than 30 A, lead to the conclusion that varying the medial [4Fe-4S] cluster potential over a 100 mV range does not have a significant effect on the inherent kinetics of electron transfer to and from the active-site flavin. The results support a proposal that the reduction potential of an individual electron relay site in a multicentered enzyme is not a strong determinant of activity; instead, as deduced from the potential dependence of catalytic electron transfer, electron flow through the intramolecular relay is rapid and reversible, and even uphill steps do not limit the catalytic rate.

The Escherichia coli complex II homologues succinate:ubiquinone oxidoreductase (SQR, SdhCDAB) and menaquinol:fumarate oxidoreductase (QFR, FrdABCD) have remarkable structural homology at their dicarboxylate binding sites. Although both SQR and QFR can catalyze the interconversion of fumarate and succinate, QFR is a much better fumarate reductase, and SQR is a better succinate oxidase. An exception to the conservation of amino acids near the dicarboxylate binding sites of the two enzymes is that there is a Glu (FrdA Glu-49) near the covalently bound FAD cofactor in most QFRs, which is replaced with a Gln (SdhA Gln-50) in SQRs. The role of the amino acid side chain in enzymes with Glu/Gln/Ala substitutions at FrdA Glu-49 and SdhA Gln-50 has been investigated in this study. The data demonstrate that the mutant enzymes with Ala substitutions in either QFR or SQR remain functionally similar to their wild type counterparts. There were, however, dramatic changes in the catalytic properties when Glu and Gln were exchanged for each other in QFR and SQR. The data show that QFR and SQR enzymes are more efficient succinate oxidases when Gln is in the target position and a better fumarate reductase when Glu is present. Overall, structural and catalytic analyses of the FrdA E49Q and SdhA Q50E mutants suggest that coulombic effects and the electronic state of the FAD are critical in dictating the preferred directionality of the succinate/fumarate interconversions catalyzed by the complex II superfamily. . The membrane-extrinsic domain is bound to the membrane through interactions with the hydrophobic subunits of the complex. These subunits comprise two membrane anchor polypeptides, each containing three transmembrane helices and providing a binding site(s) for quinone (for reviews, see Refs. 5-8). In addition, the E. coli SQR hydrophobic peptides bind one b-type heme, whereas the E. coli QFR lacks heme.Comparison of the structures of complex II is possible due to the availability of x-ray crystallographic structures for both SQR and QFR of E. coli (9, 10), the porcine SQR (11), the QFR from Wolinella succinogenes (12), and soluble homologs of the flavoprotein subunit that function as periplasmically localized fumarate reductases (13-16). The flavoprotein subunits from the E. coli SQR and QFR are highly homologous, with 64% similarity and 44% identity of amino acid residues (3). The sequence similarity within the SQR/QFR superfamily is reflected in structural alignments of members whose structures are known (17)(18)(19). Within this group, the backbone C ␣ atoms can be superimposed with a maximal root mean square deviation of 1.5 Å (10). A detailed hydride transfer mechanism for fumarate reduction has been proposed based on structural data and enzyme assays of wild type and mutant enzymes (15, 18 -20). QFRs and SQRs all contain covalently bound FAD and are bidirectional (i.e. they will catalyze both succinate oxidation and fumarate reduction). There are, however, significant differences for the kinetics of fumarate reduction by...

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...

The multi-subunit mammalian NADH-ubiquinone oxidoreductase (complex I) is part of the mitochondrial electron transport chain and physiologically serves to reduce ubiquinone with NADH as the electron donor. The three-dimensional structure of this enzyme complex remains to be elucidated and also little is known about the physiological regulation of complex I. The enzyme complex in vitro is known to exist as a mixture of active (A) and de-active (D) forms [Biochim. Biophys. Acta 1364 (1998) 169]. Studies are reported here examining the effect of anoxia and reperfusion on the A/D-equilibrium of complex I in rat hearts ex vivo. Complex I from the freshly isolated rat heart or after prolonged (1 h) normoxic perfusion exists in almost fully active form (87+/-2%). Either 30 min of nitrogen perfusion or global ischemia decreases the portion of active form of complex I to 40+/-2%. Upon re-oxygenation of cardiac tissue, complex I is converted back predominantly to the active form (80-85%). Abrupt alternation of anoxic and normoxic perfusion allows cycling between the two states of the enzyme. The possible role in the physiological regulation of complex I activity is discussed.