Succinate as Donor; Fumarate as Acceptor
THOMAS M. TOMASIAK,1 GARY CECCHINI,2* AND TINA M. IVERSON1*
[SECTION EDITOR: VALLEY STEWART]
Posted August 13, 2007
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-6600,1 and Molecular Biology Division, VA Medical Center, San Francisco, CA 94121, and Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 941432
*Corresponding authors. Mailing addresses: for Tina M. Iverson, Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-6600. Phone: (615) 322-7817, Fax: (615) 343-6532, E-mail:
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; for Gary Cecchini, Molecular Biology Division, VA Medical Center, San Francisco, CA 94121. Phone: (415) 221-4810 (ext. 4416), Fax: (415) 750-6959, E-mail:
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In respiration, organisms catalyze diverse oxidation-reduction reactions, first, to establish a transmembrane electrochemical gradient, and second, to use this energy to form ATP with the ATP synthase (77). Aerobic respiration uses O2 as the terminal electron acceptor (Fig. 1a) and is the most energy-efficient respiration pathway (108). Anaerobic respiration can use a variety of small inorganic and organic molecules as terminal electron acceptors, although these pathways result in lowered ATP levels for the cell. Compounds utilized include sulfate (13), nitrate (39, 40), nitrite (53), and organic acceptors such as fumarate (Fig. 1b) (113). In Escherichia coli, aerobic and anaerobic respiration converge on the chemical interconversion of succinate and fumarate. Succinate (Fig. 2a) is used as an electron donor during aerobic respiration (by oxidation to fumarate), while fumarate (Fig. 2b) is reduced to succinate during anaerobic respiration as a terminal electron acceptor (35, 108). In E. coli the oxido-reduction of fumarate and succinate is linked to a second oxidation-reduction reaction of the membrane-soluble small molecule quinones (Fig. 3). Since quinones are membrane embedded, the respiratory complexes coupling succinate-fumarate interconversion to quinone-quinol interconversion are integral membrane proteins. It was previously demonstrated that two distinct enzymes interconvert fumarate and succinate in E. coli (35). Succinate:quinone oxidoreductase (SQR, also termed succinate dehydrogenase or Complex II in mitochondria) is an essential component of the tricarboxylic acid cycle in aerobically grown prokaryotic and eukaryotic cells, in which it oxidizes succinate to fumarate coupled to the reduction of ubiquinone. The enzyme is encoded by the sdhCDAB operon (Fig. 4). During anaerobic respiration, in many prokaryotes and lower eukaryotes, the structurally related enzyme quinol:fumarate reductase (QFR, also termed fumarate reductase) oxidizes menaquinol in the membrane domain and reduces fumarate to succinate in the cytoplasm. QFR is also encoded by a compact operon, but the gene order is frdABCD (Fig. 4).
E. coli, as a facultative anaerobe, grows readily in aerobic, microaerophilic, or anaerobic environments. To accomplish this feat, the organism has developed an elaborate mechanism to control expression of metabolic enzymes used in catabolic and anabolic pathways, mainly at the transcriptional level (95, 96, 100). The transcription of both the SQR and QFR respiratory complexes is controlled by the availability of O2 (42, 49, 61). SQR is highly expressed in the presence of O2, whereas QFR is expressed under anaerobic or microaerophilic conditions. As a result, the level of one enzyme is usually predominant, depending on growth conditions. SQR levels rise in the presence of oxygen due to deactivation of the repressor ArcB (42) by the oxygen-sensitive kinase ArcA (43) (see Global Control of Respiratory Enzyme Synthesis). In contrast, the fumarate-nitrate reductase regulator (FNR) controls QFR levels (56) by forming a dimer (61) that activates transcription when oxygen levels fall (see Global Control of Respiratory Enzyme Synthesis and C4-Dicarboxylate Degradation in Aerobic and Anaerobic Growth).
During aerobic respiration, SQR oxidizes succinate to fumarate (succinate/fumarate couple Εm7= +30 mV [32]) and transfers two electrons and two protons to ubiquinone (Εm7 UQ/UQH2 = +90 mV [84]), reducing it to ubiquinol (UQH2). Both ubiquinone and ubiquinol are freely diffusible within the membrane. After dissociation of UQH2 from SQR, the quinol is transferred to downstream oxidases (12, 91) (see Chapter Respiration of Nitrate and Nitrite) for more details), where reoxidation of quinol by the cytochrome bd or bo3 quinol oxidases provides input electrons for further downstream reactions. Transmembrane oxido-reduction of quinones is also a general mechanism of generating a transmembrane electrochemical potential (77); however, the oxidation of succinate by SQR does not result in any charge separation across the membrane, and so SQR is not a proton pump and enzyme activity is not affected by membrane potential (Fig. 5). It has been suggested that this is the case in both E. coli and mitochondrial SQR because the electron transfer reactions are not sufficiently exergonic to promote proton translocation (102). There are cases of di-heme SQRs, such as that from Bacillus subtilis, which use menaquinone as a substrate and do not undergo endergonic electron transport that responds to the membrane proton or electrochemical potential (99). These di-heme SQRs are found in gram-positive bacteria, are succinate-menaquinone oxidoreductases, and are classified differently than E. coli SQR. Their properties have been reviewed elsewhere (32, 57, 65).
QFR catalyzes the reverse reaction of SQR, namely, the reduction of fumarate to succinate (succinate/fumarate couple Εm7 = +30 mV [32]) coupled to menaquinol (Εm7 MQ/MQH2 = −74 mV) oxidation (108, 113). This reaction is the final step in anaerobic respiration with fumarate. During anaerobic respiration with fumarate, the transmembrane proton gradient is established by respiratory complexes upstream of QFR, such as formate dehydrogenase (120), hydrogenase (50), and glycerol-3-phosphate dehydrogenase (55, 76) (see Chapters The Aerobic and Anaerobic Respiratory Chain of Escherichia coli and Salmonella enterica: Enzymes and Energetics) and Oxygen as Acceptor). The contribution that QFR makes to the transmembrane gradient is thus indirect since it couples the reoxidation of MQH2 to MQ to fumarate reduction (35, 55). This generates oxidizing equivalents for other respiratory complexes (59). As for E. coli SQR, this is most easily explained by the fact that the fumarate reduction site is in the cytoplasm and the menaquinol oxidation site is at the membrane cytoplasmic interface. Thus, protons released by menaquinol oxidation and fumarate reduction balance each other and remain in the cytoplasm and no net proton gradient is produced. The E. coli QFR is different in this aspect from di-heme-containing QFRs, such as those from Wolinella succinogenes, as discussed in a series of interesting articles from the Lancaster laboratory (55, 57, 65).
Despite opposite catalytic directions under normal physiological conditions, QFR and SQR have similar molecular weights (20, 22, 24, 63, 119) and architecture (44, 121), contain nearly identical sets of cofactors, can catalyze succinate-fumarate interconversion bi-directionally in vitro (Table 1), and can functionally replace one another in vivo (31, 66). SQR and QFR contain four nonidentical subunits, and the enzymes can be divided into hydrophilic and hydrophobic domains (Fig. 4). The hydrophilic subunits A and B of both enzymes are sufficient for the succino-oxidase/fumarate reductase activities of the enzymes with artificial electron acceptors. These domains are often referred to as the succinate dehydrogenase and fumarate reductase portions of SQR and QFR, respectively. The hydrophobic membrane anchor domain subunits C and D are necessary to confer the quinone oxidoreductase activity to both SQR and QFR. Although both enzymes are bi-directional in that they readily oxidize succinate or reduce fumarate, they are poised to function differently in vivo. As is shown in Table 1, SQR—in catalytic assays using artificial electron donors/acceptors—is 50-fold more efficient as a succino-oxidase than it is as a fumarate reductase. Conversely, QFR is only fivefold more efficient as a fumarate reductase than it is as a succino-oxidase. The enzymes are, however, physiologically quinone oxidoreductases, but the situation is a little more complicated in trying to directly compare catalytic activity for the enzymes with quinones (67). Nevertheless, SQR is more efficient at reducing ubiquinone than is QFR, whereas QFR is much more efficient at oxidizing menaquinol than is SQR (15, 67). Thus, not surprisingly, the enzymes appear to have evolved to function best in their proper ecological niche.
Table 1Catalytic parameters for succinate oxidase and fumarate reductase reactions catalyzed by E. coli SQR and QFR |
Both SQR and QFR from E. coli are hetero-tetrameric membrane proteins with two subunits (sdhA, frdA, flavoprotein subunits; sdhB, frdB, iron-sulfur proteins) forming an extra-membrane domain and two subunits (sdhC, cyt bL [SQR] frdC; and sdhD, cyt bS [SQR], frdD) forming an integral-membrane domain (residue numbers retain the chain name to prevent confusion). This architecture is optimally designed to couple the oxido-reduction of a water-soluble dicarboxylate to a membrane-soluble quinone. Spectroscopic and structural studies have revealed the presence of four covalently attached cofactors in each complex: a flavin adenine dinucleotide (FAD) (51) covalently linked to a histidyl residue, and three iron-sulfur (Fe:S) clusters. The covalent linkage of the FAD cofactor to the mammalian mitochondrial SQR was the first example of such a linkage to any protein (114), and it was subsequently shown that E. coli QFR contained a similar linkage (116). After many years of controversy in the 1980s it became clear that three distinct types of iron-sulfur clusters are present in both mammalian and prokaryotic SQR and QFR. The history of this controversy is elegantly reviewed by Beinert (5), and this article includes many of the primary references for determination of the Fe:S composition of SQR and QFR. In the case of eukaryotic and E. coli SQR and QFR the Fe:S clusters include a [2Fe:2S]2+,1+ cluster, a [4Fe:4S]2+,1+ cluster, and a [3Fe:4S]1+,0 cluster (Fig. 6). It is gratifying that the initial crystal structures of the E. coli enzymes (44, 121) confirmed that the assignments for the Fe:S clusters were correct. In addition to the covalent flavin and Fe:S centers, SQR (but not E. coli QFR) contains a noncovalently bound b-type heme in the membrane-spanning region (52). In both enzymes, the FAD located in subunit A is part of the active site for dicarboxylate interconversion, and an active site for the interconversion of Q and QH2 lies at the cytoplasmic side of the membrane interface and is composed of residues from the B, C, and D subunits (44, 121). Analyses of the X-ray structures (Fig. 4) also indicated that the cofactors were arranged linearly with intercofactor distances <14 Å (Fig. 7) within the physiological limit of electron transfer reactions (86). As shown in Fig. 4, the structures also confirm other results from spectroscopic and enzymatic studies, notably the overall similarity between SQR and QFR. As anticipated, SQR and QFR each adopt similar folds in the soluble domain. Structural alignment of the flavoprotein subunit of E. coli SQR and QFR shows a root mean square (rms) deviation of 1.44 Å for 535 Cα atoms, while structural alignment of the iron-sulfur protein reveals an rms deviation of 1.256 Å for 221 Cα atoms.
The flavoprotein is the largest subunit in each enzyme: 64 kDa in SQR and 67 kDa in QFR (20, 119). Of the four subunits in SQR and QFR, the flavoprotein exhibits the highest sequence identity between the two enzymes (41% identity and 58% similarity). The flavoprotein contains a covalently linked FAD (114, 116, 121), which is at the active site for dicarboxylate interconversion. This covalent linkage is formed between the Nε atom of an absolutely conserved histidine (His-A45, SQR; His-A44, QFR) and the C8 atom of the FAD (Fig. 8) ( 114, 116).
Formation of the histidyl-FAD bond presumably is autocatalytic in SQR and QFR but requires catalytic turnover of the enzyme to complete the covalent linkage. Proposed mechanisms for the covalent attachment of flavins to proteins have been reviewed in detail (75), and the architecture of the FAD binding site in SQR and QFR is consistent with the autocatalytic iminoquinone methide attachment (27, 28). Thus, mutation of side-chain amino acids that affect binding of dicarboxylate substrates to the active site often results in loss of covalent linkage to FAD, as was initially shown with QFR (10). Mutations that affect the covalent FAD linkage in human SQR have been linked to late-onset neurodegenerative disease (9), and the equivalent mutations in the E. coli enzymes (sdhA Arg399 or frdA Arg390) are also known to result in enzyme containing noncovalent FAD (16). Covalent attachment of FAD is one factor, along with the protein environment, that results in a dramatic increase in the reduction potential of the covalent FAD compared with free FAD (Εm7 = −220 mV) to ~−70 mV in SQR and ~−55 mV in QFR (2, 10, 26, 89). The rise in reduction potential along with other electronic factors affecting the flavin becomes crucial for succinate oxidation (10, 26, 75). Site-directed mutants of either SQR or QFR that lose covalent linkage to FAD can no longer oxidize succinate but retain fumarate reductase ability (10). Soluble homologs of the flavoprotein subunit exist in several obligate anaerobes (79, 88); these fumarate reductase enzymes contain a noncovalent FAD with a lower reduction potential (79, 92) and are essentially unable to oxidize succinate (92). The evolutionary retention of a covalently attached FAD in QFR may reflect the in vivo requirement for QFR to oxidize succinate when E. coli is switching between anaerobic and aerobic respiration.
The mechanism of succinate-fumarate interconversion in SQR and QFR.
The interconversion of succinate and fumarate occurs at the dicarboxylate binding site of SQR and QFR, as described below. Initial studies of succinate oxidation used preparations of succinate dehydrogenase that were not pure, and it was not initially recognized that the complete SQR enzyme was membrane bound. Nevertheless, it was shown that mammalian succinate dehydrogenase was a reversible enzyme capable of both succino-oxidase and fumarate reductase activities (71). Succinate dehydrogenase or fumarate reductase activity is most conveniently measured using artificial electron acceptor/donors of appropriate redox potential (3, 69, 88). Succinate oxidation by both succinate dehydrogenase and fumarate reductase can be spectrophotometrically assayed using primary electron acceptors such as phenazine ethosulfate (or phenazine methosulfate) (3) coupled with the reduction of 2,6-dichlorophenolindophenol. Fumarate reduction by either enzyme can be assayed using reduced viologen dyes such as benzyl or methyl viologen (3). Note that, as isolated, succinate dehydrogenase and fumarate reductase contain tightly bound inhibitory oxaloacetate (OAA) at their dicarboxylate binding site (3). The OAA binds more tightly to the oxidized forms of the enzyme than it does to the reduced enzymes (2, 3). Thus, to accurately measure the initial rate of succinate oxidation the OAA must be removed from the dicarboxylate binding site by "activating" the enzyme in the presence of succinate or other weaker binding dicarboxylates, such as malonate (2, 3). Since the OAA binds much more weakly to reduced succinate dehydrogenase and/or fumarate reductase, it is not necessary to activate the enzymes to measure fumarate reduction.
The dicarboxylate-interconversion reaction mechanism has been best studied in the fumarate reduction direction by using soluble homologs of fumarate reductase from Shewanella spp. (80, 92). In E. coli QFR, as in its soluble homologs, fumarate reduction proceeds in two steps (Fig. 9). In the first step, the reduced FAD transfers a hydride to the double bond of fumarate, forming a carbanion intermediate. The hydride transfer is likely to be the rate-determining step (64). In the second step, the carbanion intermediate accepts a proton from Arg-A287 (Fig. 9).
Several highly conserved residues, including His A-242 (SQR)/His-A232 (QFR), Glu-A255 (SQR)/Glu-A245 (QFR), Arg A-286 (SQR)/Arg-A287 (QFR), His A-354 (SQR)/His-A355 (QFR), and Arg A-399 (SQR)/Arg-A390 (QFR), line the active site (Fig. 10). An elegant combination of site-directed mutagenesis (25, 80, 87, 112) and crystallography (107) in the soluble fumarate reductase homologs assigned Arg-A287 (QFR) as the proton shuttle for the second step of the reaction, while the remaining conserved residues are likely responsible for binding of the substrate. A number of secondary proton shuttles may provide a pathway for protons between the active site and the cytoplasm (Fig. 9) (112).
The local active-site environment facilitates fumarate reduction in many ways. First, hydrogen bonds from Arg-A390 and His-A355 to fumarate create a dipole along the fumarate double bond (92). The electrostatic effect induces a δ+ charge on the C3 carbon of the fumarate double bond, making fumarate more susceptible to hydride transfer at this position. Second, twisting of the fumarate double bond (Fig. 11) by the protein environment may destabilize π-π overlap and raise the fumarate free energy. Structures cocrystallized with fumarate show a twisted conformation for the C2-C3 double bond (60). Domain rearrangements near the flavoprotein active site may stabilize the twisted conformation to facilitate catalysis by forcing fumarate to mimic the transition state (58, 92).
In classical enzymology, the enzyme provides an alternate path to product formation, speeding up the reaction in both the backward and forward direction. SQR and QFR violate the definition of a classic catalyst. While each enzyme can catalyze the opposite reaction in vitro and can functionally replace the other in vivo (31, 66), both enzymes are more efficient in one direction than in the other (Table 1). So far, two separate pieces of the molecular architecture have been shown to control the direction of the reaction. The first is the identity of the side-chain residues near the electron transfer cofactors of each enzyme. These side chains tune the reduction potentials of each cofactor and control the preferential direction of electron transfer through the complex (Fig. 7). In considering the full fumarate reduction reaction 2H+ + 2e− + fumarate → succinate, it is clear that electrons are one substrate. Since SQR limits the availability of electrons at the active site, fumarate reduction is not preferentially catalyzed, even in the presence of fumarate, due to limitation of the electron substrate.
A second parameter controlling the direction of the reaction in SQR and QFR is control of the one-electron reduced flavin semiquinone intermediate during enzyme turnover. Electron paramagnetic resonance (EPR) analysis of SQR and QFR reveal differences in the FAD flavin semiquinone intermediate during enzyme turnover, with the SQR FAD transitioning through anionic flavin semiquinone (FAD•−), while QFR apparently uses a neutral flavin semiquinone (FADH•) (69).
The side chains within coulombic distance of the FAD may suggest how the flavin semiquinone intermediate is controlled. An examination of the environment around the FAD in the E. coli SQR and QFR, as well as other homologs, reveals an identical residue at a conserved position depending on whether the enzyme is SQR or QFR. SQR normally has a glutamine residue (Q50 in E. coli SQR), while the preponderance of QFRs have a glutamate (E49 in E. coli QFR) at the equivalent position. When the equivalent residues are swapped between SQR and QFR (i.e., the glutamine in SQR is switched to a glutamate like that found in QFR and the converse mutation is done to QFR), SQR becomes a better fumarate reductase while QFR becomes a more efficient succinate oxidase (69). This is explained, in part, by the observation that electrostatic differences caused by the γ-carboxylate of glutamate or the amide of glutamine shift the redox potential of FAD and the stability of the flavin semiquinone radical so that the radical in QFR becomes more like that in SQR (37, 69). The change in reduction potential along with the changed stabilization of the flavin radical in part accounts for kinetic differences between both enzymes and suggests that there is a change in the protonation state of the FAD environment. In addition, these studies suggest that the "forward reaction" of each enzyme uses a hydride transfer while the "backward reaction" uses a hydrogen atom transfer. These differences are not enough, however, to account for all of the catalytic changes observed that probably reflect a multitude of subtle changes in the flavin environment and dicarboxylate binding site (69).
The above observations may relate to another intriguing catalytic difference between SQR and QFR in their respective abilities to reduce fumarate. As with most enzymes, fumarate reductase increases its rate of catalysis when there is a greater thermodynamic driving force, but succinate dehydrogenase demonstrates an unusual behavior termed the "tunnel diode" effect (105). These studies were initially done with the mitochondrial succinate dehydrogenase; however, the E. coli enzyme shows similar properties (89). It was shown that succinate dehydrogenase is able to efficiently catalyze fumarate reduction above a redox potential of approximately −60 mV (7.0); conversely, below this potential, the rate of catalytic fumarate reduction abruptly decreases even though the redox driving force has increased. This effect resembles the current-voltage characteristics of a tunnel diode found in electronic circuits. Succinate dehydrogenase is an excellent fumarate reductase, yet this activity is only found over a narrow range of redox potential. Note that fumarate reductase does not demonstrate this behavior, as its activity increases along with its potential driving force. The physical principles underlying the tunnel diode effect have not been determined but have been suggested to relate to subtle differences in the properties of the dicarboxylate binding site of SQR and QFR (89, 105). It is possible that the tunnel diode effect may limit fumarate reduction under hypoxic conditions and prevent SQR reduction of fumarate during anoxic conditions.
The iron-sulfur protein subunit comprises two distinct domains and is the second largest subunit (26.8 kDa in SQR and 27.0 kDa in QFR). The first domain adopts a plant-type ferredoxin fold (type I ferredoxin) and contains the [2Fe:2S] cluster, while the second domain adopts a bacterial (type II ferredoxin) ferredoxin fold and contains both the [3Fe:4S] and the [4Fe:4S] clusters (Fig. 12) (44). The iron protein is physically positioned between the dicarboxylate interconversion active site in the flavoprotein subunit and the Q—QH2 interconversion active site in the membrane. The Fe:S clusters provide a pathway for efficient electron transfer between the two active sites of these enzymes.
The history of the discovery of Fe:S clusters in SQR and QFR is described in an excellent review from Beinert (5). Note that succinate dehydrogenase was one of the first proteins known to demonstrate tightly bound iron (104). The compositions of the Fe:S clusters of SQR were first identified and characterized with EPR spectroscopy in bovine heart mitochondria (6, 7, 85, 94). Subsequent studies in E. coli QFR confirmed the same composition of Fe:S clusters in this enzyme (47, 48, 78). In mitochondrial SQR, E. coli SQR, and QFR, Fe:S cluster irons are coordinated by cysteines in ferredoxin-like sequence motifs with the [4Fe:4S] cluster coordinated by a C-X-X-C-X-X-C-X3-C-P motif and the [3Fe:4S] cluster coordinated by a C-X5-C-X3-C-P motif (22, 24). In QFR, the [2Fe:2S] cluster is coordinated by a C-X4-C-X-X-C-X10-C-P motif. Infrequently, in E. coli SQR, the third cysteine of the [2Fe:2S] coordinating motif is replaced by an aspartate (24), a unique alteration of this motif in SQRs. Mutation of Cys-B65 in QFR to aspartate to mimic the SQR [2Fe:2S] structure had no overall effect on the midpoint potential or catalytic activity (117), and it is not known why aspartate is found in this position in SQR (Fig. 6).
As mentioned above, the reduction potential of the Fe:S clusters is tuned to control the availability of electrons at the active site FAD (Fig. 7). All three Fe:S clusters of SQR have higher midpoint potentials (83, 85) than their counterparts in QFR, but in both enzymes, the reduction potentials of the [4Fe:4S] cluster is unusually low. Prior to the determination of crystal structures, it was speculated that this Fe:S cluster could be off-pathway for electron transfer (54). The crystal structure reveals that edge-to-edge distances of the [4Fe:4S] cluster to the [2Fe:2S] and [3Fe:4S] clusters (Fig. 7) fall within physiological electron transfer ranges (<14Å) (86). While the thermodynamic barrier imposed by the low [4Fe:4S] cluster potential suggests that electron transfer to the [4Fe:4S] cluster is not rate limiting, site-directed mutagenesis of E. coliSQR that introduced negative charges near the 4Fe:4S lowered the potential (by ~70 mV to −285 mV) and decreased catalytic turnover by 65% (17). That the [4Fe:4S] cluster of QFR is a direct participant in electron transfer reactions had previously been shown by protein film voltammetry and mutagenic studies of E. coli QFR (37). These studies showed that when the [4Fe:4S] cluster is reduced it results in a boost in catalytic rate for fumarate reduction by QFR. This is somewhat reminiscent of changes in the electronic properties of the FAD that apparently contribute to the tunnel diode properties of SQR in the flavoprotein subunit. These findings are consistent with an interpretation in which oxido-reduction of the redox centers has direct influence on catalytic rates in both SQR and QFR.
The integral membrane subunits of SQR and QFR contain the second active site in the enzyme (Q-site), which performs oxido-reduction of the membrane-soluble small molecule quinone. In contrast to the soluble subunits of SQR and QFR, which share considerable sequence similarity, there is no detectable sequence identity in the integral membrane subunits that contain the Q-site. Despite this, the C and D integral membrane subunits of both enzymes each comprise three transmembrane helices that come together to form a four-helix bundle structural motif (Fig. 13).
The membrane-spanning subunits of SQR and QFR additionally differ in cofactor association. SQR contains a b-type heme, heme b556, which is absent in QFR (Fig. 14). Each complex also preferentially associates with a different type of quinone molecule; in SQR, ubiquinone (Fig. 3b) is used as the physiological electron acceptor, while in QFR menaquinol (Fig. 3c) is used as the electron donor. Note, however, that the Q-binding sites in both QFR and SQR can accommodate either ubiquinone or menaquinone molecules as evidenced by both in vivo and in vitro studies (31, 66, 69). One explanation of why the enzymes preferentially bind one quinone over the other is that the redox potential of the [3Fe:4S] cluster, which is the direct electron donor/acceptor for SQR and QFR, respectively, is poised to interact preferentially with either the higher potential ubiquinone (+ 90 mV) or the lower potential menaquinone (−74 mV). As would be expected for active sites that bind different small molecules, the side-chain residues of each Q-site differ to optimize the binding of one species (Fig. 15). The location of the Q-site with respect to the soluble domain also differs; an alignment of SQR and QFR soluble domains results in Q-sites that are positioned 15 Å away (Fig. 14) from each other (121).
Ubiquinone binding in SQR.
It was shown by EPR spectroscopy more than 30 years ago that mitochondrial SQR stabilizes a ubi-semiquinone radical (94). Photoaffinity labeling with quinone analogs also identified potential amino acid residues in bovine mitochondrial SQR involved in side-chain interactions with the quinone (62, 103). The crystal structure of E. coli SQR (Fig. 15) confirmed the general architecture of the quinone binding site in bacterial SQR (121). In E. coli SQR both the crystal structure (89) and kinetic data (67) are consistent with a single catalytic pocket termed the QP-site (for Q-proximal), where quinone binds. The QP-site is located within 8 Å of the [3Fe:4S] cluster and 7 Å from the edge of the b-heme moiety, both distances well within the range for efficient electron transfer (86). Co- crystal structures are available for the E. coli SQR with ubiquinone and the Q-site inhibitor atpenin-A5 (36). The different binding positions for these molecules suggest that ubiquinone and ubi-semiquinone (the one-electron intermediate) bind in the SQR Q-site in two distinct positions during the catalytic cycle, consistent with other quinone binding sites, suggesting a general plasticity for quinone binding sites. This phenomenon has previously been observed in the photosynthetic reaction center (19) and is proposed as a mechanism to protect the ubi-semiquinone radical intermediate from interaction with solvent.
The costructure of SQR with ubiquinone (121) reveals a single hydrogen bond between the ubiquinone O1 atom and the side chain hydroxyl of Tyr-D83 (Fig. 15). The costructure with the Q-site inhibitor atpenin-A5 (36) reveals that a deeper binding pocket for quinone is available (Fig. 16), which would prevent the premature disassociation of a highly reactive, partially reduced semiquinone. In the second binding position, additional hydrogen bonds to Ser-C27 and His-B207 bring ubiquinone into close proximity to a putative proton shuttle pathway (Fig. 17). This putative pathway would provide a mechanism for protons to enter the binding pocket in the membrane-spanning region.
Cyt b556.
The crystal structure of SQR (121) includes a b-type heme (52) that uses His-C84 and His-D71 as the Fe-ligands, confirming what was expected from spectroscopic studies (23, 70, 90). The position of heme b556 with respect to the electron transfer pathway was a surprise since the heme is off-pathway (Fig. 7) for electron transfer between the [3Fe:4S] cluster and ubiquinone. As electrons are delivered to the Q-site from the [3Fe:4S] cluster, the structure raises the possibility that the first electron can reduce either the heme directly or the quinone. It is ~11 Å from the [3Fe:4S] cluster to the edge of the heme, whereas it is only ~7 Å from the Fe:S cluster to the quinone (121). Subsequently, EPR studies indicate that an intact quinone binding site is necessary for reduction of the heme (111). Although the logical conclusion from these studies is that the quinone is reduced before the heme, pulse radiolysis investigation of isolated E. coli SQR suggests that there can be a very rapid equilibration of the electron between the heme and the quinone so that first electron could be delivered to either redox center (4). Further study is clearly needed to clarify the role order of delivery of electrons to the b-heme.
The overall role of the heme in SQR is also controversial. One role for the heme is structural, as it has been shown that assembly of the SQR holoenzyme is perturbed in E. coli heme-deficient mutants (82). Another proposal is that the heme may act as an "electron sink" during reduction of the enzyme. This additional cofactor would prevent electrons from accumulating on the FAD when ubiquinone is not immediately available by diverting electrons away from the iron-sulfur clusters (121). Since single-electron-reduced FAD is highly reactive, an additional cofactor in the membrane-spanning region would prevent back-reactions in vivo (i.e., fumarate reduction) and, more importantly, would prevent inappropriate reactions such as the partial reduction of O2 to form harmful reactive oxygen species (ROS) during aerobic respiration. The redox potentials of the cofactors in SQR would tend to pull electrons away from the flavin site, which is in direct opposition to what is the case for E. coli QFR where the cofactors are tuned so electrons are transferred toward the FAD (Fig. 7). This proposal is consistent with the known tendency of QFR to form ROS in vitro when oxygen is available from the flavin binding site, while SQR does not (38, 74). These interesting findings suggest one reason that organisms have evolved both a SQR and a QFR. As pointed out by Imlay (38), as E. coli transits from an anaerobic environment where QFR is highly expressed to an aerobic one, the ROS produced by QFR could prove deleterious to the cell. SQR, which produces much less ROS than QFR, would thus be the preferred enzyme for cells in a more aerobic environment (38).
Menaquinol binding in QFR.
The structure of QFR revealed electron density attributed to menaquinol in two positions: a proximal QP-position located in proximity to the [3Fe:4S] cluster of the frdB iron-sulfur protein subunit and a distal QD-position located on the opposite side of the membrane (Fig. 14) (44). The QP- and QD-sites are separated by ~25 Å, a distance too far for physiological electron transfer (86), in part explaining why QFR cannot itself support formation of a proton gradient. While the relevance of QD continues to be investigated, it is clear that menaquinol binding the QP-site is the immediate electron donor to the [3Fe:4S] cluster (32, 68, 106). Note also that a recent X-ray structure of porcine SQR also revealed two binding sites for the quinone site inhibitor 2-thenoyltrifluoroacetone (TTFA), and these sites were spatially separated across the membrane in a manner similar to what was seen in QFR (106). There is no evidence that this distal quinone binding site has any functional significance in the mammalian SQR, and the hydrophobic TTFA inhibitor was crystallized with the enzyme at a concentration some two orders of magnitude higher than necessary for inhibition of the enzyme. It is also relevant that the di-heme members of the SQR and QFR family of enzymes (32, 57, 59, 60, 72) have a functional quinone catalytic site on the distal side of the membrane. Thus, it should be considered that the QD-site might be an evolutionarily conserved hydrophobic pocket that is functionally active in the di-heme-containing enzymes but nonfunctional in the single heme and/or no-heme members of the SQR/QFR family.
Unlike SQR, where quinone analogs adopt somewhat different conformations as compared to ubiquinone, comparisons of the costructure of QFR and menaquinol with the costructure of QFR and the semiquinone analog 2-N-heptyl-4-hydroxyquinoline N-oxide (HQNO) (45) reveal that both menaquinol and the semiquinone analog effectively bind to the same site (Fig. 16). Several residues in the membrane-spanning subunits stabilize menaquinol binding at this position, including Trp-D14, Arg-C28, and Glu-C29 (Fig. 15) (44). Glu-C29 had previously been identified in random mutagenesis studies as being important for quinol binding (118). EPR analysis of the Glu-C29 → Leu mutation showed an alteration in the stability of the semiquinone radical by increasing the lifetime by four orders of magnitude (33). Glu-C29 may play an important catalytic role in protonation reactions and by destabilizing the semiquinol radical to prevent possible side reactions between it and oxygen.
An unusual aspect of the Q-site in QFR is the formation of a hydrogen-bonding interaction from the Lys-B228 Nζ atom to the menaquinol O1 atom (Fig. 15) (45). Lys-B228 lies on an amphipathic helix that rests on the membrane, and the side chain is inserted down into the membrane bilayer. The formation of a hydrogen-bonding interaction with menaquinol would neutralize the positive charge of this side chain and additionally suggests that this side chain is a possible proton donor (45). Even a conservative mutation to arginine abolishes both menaquinol oxidation and ubiquinone reduction.
As is described above, SQR and QFR share sequence similarity in the soluble domain, which suggests that they evolved from a gene duplication event. The strongest sequence similarity (58%) is observed in the flavoprotein subunit (A chain), which contains the active site for succinate-fumarate interconversion. The flavoprotein subunit also exhibits significant sequence and structural similarity to soluble fumarate reductases, as well as to L-aspartate oxidase in E. coli. These soluble counterparts lack a covalent bond to FAD and consequently lack succinate-oxidizing ability (8, 79, 81, 109). This suggests that fumarate reduction came about first, with the ability to oxidize succinate occurring later with the formation of a covalent bond to FAD. Sequence comparisons between mitochondrial SQR and eubacterial homologs predict that those lineages have an ancient common origin in α-proteobacteria (11, 101).
Evidence for an ancient evolutionary origin of complex II enzymes comes from sequence relationships between iron-sulfur protein subunits (subunit B) of different species (14). The iron-sulfur protein subunit is related in sequence to ferredoxins (Fig. 12), evolutionarily ancient redox-active proteins present in prokaryotes, in mitochondria and chloroplasts of eukaryotes, and in archaea. This suggests that they may have been present in the last universal common ancestor (LUCA) as part of a diverse array of respiratory processes. The iron-sulfur protein subunit originally may have been a separate soluble protein that transferred electrons between divergent reductases and membrane-bound four-helix bundle cytochromes. Over time, the iron-sulfur protein developed affinity for the flavoprotein subunit, followed by the membrane-spanning subunits, leading to the current subunit arrangement. Sequence and cofactor differences in the integral membrane subunits (subunits C and D) place SQR and QFR into separate subclasses (32), and here the enzymes differ substantially. E. coli SQR contains two transmembrane polypeptide chains bound to a single heme. In contrast, E. coli QFR contains two transmembrane subunits but lacks heme (32, 34). Through evolution, the iron-sulfur protein may have bound various membrane subunits, explaining the current diversity of membrane subunits found in homologs of SQR and QFR.
One unanswered question revolves around whether small- and large-scale protein movements are required for succinate-fumarate interconversion. Domain rearrangements around the flavin cofactor may provide some catalytic control in the flavoprotein subunit. The flavoprotein subunits of SQR and QFR, along with soluble fumarate reductases, contain several conserved residues near the active site that do not directly interact with the substrate. Furthermore, the conformation of two domains enclosing the active site is altered in many of the structures, which may be relevant to fumarate/succinate interconversion by coercing the molecules into a transition state and may also prevent access of water or oxygen to the active site during the formation of the transition state. The existence and importance of domain movements have been called into question since the introduction of a disulfide bond between domains, which theoretically restricts interdomain motions, does not completely abolish enzyme activity in soluble fumarate reductases (93). Furthermore, little evidence exists for how FAD is reprotonated during the active cycle or how the structure of FAD changes between oxidation and reduction.
Quinone chemistry in SQR and QFR is also an area of active interest. No well-established mechanism exists for quinone reduction in SQR or quinol oxidation in QFR. The role of the heme in electron transfer reactions in SQR and other complex II homologs remains to be established as well. Furthermore, QFR is a much more potent producer of the superoxide than is SQR, yet differences in how these enzymes produce ROS (and under what conditions) remain unknown. Higher SQR cofactor potentials and the presence of a heme may shift electrons away from FAD, the known site of ROS production in QFR.
Studies in our laboratories were supported by the Department of Veterans Affairs (G.C.) and by National Institutes of Health Grants GM61606 (to G.C.) and GM079419 (to T.M.I.), a pilot award funded by P30 ES000267 (to T.M.I.), and the Ellison Medical Foundation AG-NS-0325 (T.M.I.).
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Module3.2.6
