The high potential iron-sulfur center in Escherichia coli fumarate reductase is a three-iron cluster

The fumarate reductase complex and soluble enzyme from Escherichia coli have been investigated by low temperature magnetic circular dichroism and electron paramagnetic resonance spectroscopies. The results confirm the presence of one [2Fe-2S] cluster and show that the high potential iron-sulfur center is a 3Fe cluster of the type found in bacterial ferredoxins. Since the 3Fe cluster is present in catalytically competent enzyme and does not appear to be involved in any type of cluster conversion under reducing conditions, we conclude that it is an intrinsic component of the functional enzyme. The significance of the results is discussed in relation to the published amino acid sequence and the iron-sulfur cluster composition of bacterial fumarate reductases.     

Interactions between the molybdenum cofactor and iron-sulfur clusters of Escherichia coli dimethylsulfoxide reductase

We have used site-directed mutagenesis to study the interactions between the molybdo-bis(molybdopterin guanine dinucleotide) cofactor (Mo-bisMGD) and the other prosthetic groups of Escherichia coli Me2SO reductase (DmsABC). In redox-poised preparations, there is a significant spin-spin interaction between the reduced Em,7 = -120 mV [4Fe-4S] cluster of DmsB and the Mo(V) of the Mo-bisMGD of DmsA. This interaction is significantly modified in a DmsA-C38S mutant that contains a [3Fe-4S] cluster in DmsA, suggesting that the [3Fe-4S] cluster is in close juxtaposition to the vector connecting the Mo(V) and the Em,7 = -120 mV cluster of DmsB. In a DmsA-R77S mutant, the interaction is eliminated, indicating the importance of this residue in defining the interaction pathway. In ferricyanide-oxidized glycerol-inhibited DmsAC38SBC, there is no detectable interaction between the oxidized [3Fe-4S] cluster and the Mo-bisMGD, except for a minor broadening of the Mo(V) spectrum. In a double mutant, DmsAS176ABC102SC, which contains an engineered [3Fe-4S] cluster in DmsB, no significant paramagnetic interaction is detected between the oxidized [3Fe-4S] cluster and the Mo(V). These results have important implications for (i) understanding the magnetic interactions between the Mo(V) and other paramagnetic centers and (ii) delineating the electron transfer pathway from the [4Fe-4S] clusters of DmsB to the Mo-bisMGD of DmsA.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron–sulfur subunit which contains three distinct iron–sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b₅₅₆ is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Repair of oxidized iron-sulfur clusters in Escherichia coli

The [4Fe-4S]2+ clusters of dehydratases are rapidly damaged by univalent oxidants, including hydrogen peroxide, superoxide, and peroxynitrite. The loss of an electron destabilizes the cluster, causing it to release its catalytic iron atom and converting the cluster initially to an inactive [3Fe-4S]1+ form. Continued exposure to oxidants in vitro leads to further iron release. Experiments have shown that these clusters are repaired in vivo. We sought to determine whether repair is mediated by either the Isc or Suf cluster-assembly systems that have been identified in Escherichia coli. We found that all the proteins encoded by the isc operon were critical for de novo assembly, but most of these were unnecessary for cluster repair. IscS, a cysteine desulfurase, appeared to be an exception: although iscS mutants repaired damaged clusters, they did so substantially more slowly than did wild-type cells. Because sulfur mobilization should be required only if clusters degrade beyond the [3Fe-4S]1+ state, we used whole cell EPR to visualize the fate of oxidized enzymes in vivo. Fumarase A was overproduced. Brief exposure of cells to hydrogen peroxide resulted in the appearance of the characteristic [3Fe-4S]1+ signal of the oxidized enzyme. When hydrogen peroxide was then scavenged, the enzyme activity reappeared within minutes, in concert with the disappearance of the EPR signal. Thus it is unclear why IscS is required for efficient repair. The iscS mutants grew poorly, allowing the possibility that metabolic defects indirectly slow the repair process. Our data did indicate that damaged clusters decompose beyond the [3Fe-4S]1+ state in vivo when stress is prolonged. Under the conditions of our experiments, mutants that lacked other repair candidates--Suf proteins, glutathione, and NADPH: ferredoxin reductase--all repaired clusters at normal rates. We conclude that the mechanism of cluster repair is distinct from that of de novo assembly and that this is true because mild oxidative stress does not degrade clusters in vivo to the point of presenting an apoenzyme to the de novo cluster-assembly systems.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli.

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Assembly of Escherichia coli fumarate reductase holoenzyme

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.     

Interactions of oxaloacetate with Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA). Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents. In these respects the enzyme behaves like cardiac succinate dehydrogenase. Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized (KD approximately 0.12 microM) and reduced (KD approximately 0.9 microM) forms of fumarate reductase. Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase (B. A. C. Ackrell, E. B. Kearney, and D. Edmondson (1975) J. Biol. Chem. 250, 7114-7119), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme. However, the processes differed for the two enzymes; activation of fumarate reductase involved 2e- and 1H+, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2e- and 2H+ in cardiac succinate dehydrogenase. The reason for the difference is not known. The redox potential of the FAD/FADH2 couple in FRD (Em approximately -55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (-90 mV).     

The iron-sulfur cluster composition of Escherichia coli nitrate reductase

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.     

Iron-sulphur clusters in fumarate reductase from Vibrio succinogenes

(1) The fumarate reductase complex from Vibrio succinogenes contains one FAD molecule, one [4Fe-4S]3+(3+,2+) and one [2Fe-2S]2+(2+,1+) cluster per enzyme molecule. Both clusters can be partly reduced by succinate. In the presence of excess Na2S2O4 and fumarate, the [2Fe-2S] cluster is completely oxidized, whereas the other cluster is largely reduced. (2) The [2Fe-2S] cluster is localized in the Mr, 31,000 subunit. The EPR spectrum of the reduced cluster in the isolated subunit differs slightly in line width, but not in g-value, from the spectrum of reduced, intact enzyme complex. The demonstrates that the immediate environment of th cluster is little perturbed by dissociating this subunit from the FAD-containing Mr 79,000 subunit. The temperature dependence of the power-saturation behaviour has, however, greatly decreased in the isolated subunit, the saturation at 11 K of the paramagnetic cluster being much less than in the enzyme complex. Moreover, the temperature dependence of th power-saturation behaviour of this cluster in the enzyme is greater with succinate as reducing agent, than with dithionite. (3) The [4Fe-4S] cluster is located on the Mr 79,000 subunit. This cluster is unstable in air when the subunit has been dissociated from the enzyme complex.     

Differences in protonation of ubiquinone and menaquinone in fumarate reductase from Escherichia coli

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.     

Conformational properties of membrane-bound fumarate reductase of Escherichia coli

Anaerobically grown cells of Escherichia coli harboring the plasmid pFRD63 over-produce fumarate reductase, a membrane-bound complex localized in the inner membrane of the cell, where this enzyme represents at least 90% of the total membrane proteins (B. D. Lemire, J. J. Robinson, and J. H. Weiner (1982) J. Bacteriol. 152, 1126-1131). Preparations of inner membrane fractions suspended in 40% sucrose are optically clear, allowing optical spectroscopic measurements. Circular dichroism spectra showed that between pH 6 and 11 the secondary structure of the enzyme is at least 55% in alpha helix and that above pH 11 the structure abruptly changes to a beta-like conformation. The same phenomenon is observed in samples solubilized in the nonionic detergent C12E9. Absorption spectra of the enzyme either membrane bound or solubilized in detergents or exposed to alkaline pH showed that the accessibility of the active site to solvent components is modulated by the interaction of the protein with the membrane. Solubilization of the membrane-bound enzyme with 1% Triton X-100 or C12E9 produced a decrease in ellipticity and in enzymatic activity.     

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.     

The iron-sulfur clusters in Escherichia coli succinate dehydrogenase direct electron flow

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.     

Identification of membrane anchor polypeptides of Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli has been shown to be a membrane-bound enzyme composed of a 69,000-dalton catalytic-flavin-containing subunit and a 27,000-dalton nonheme-iron-containing subunit. Using gene cloning and amplification techniques, we have observed two additional polypeptides encoded by the frd operon, with apparent molecular weights of 15,000 and 14,000, which are expressed when E. coli is grown anaerobically on glycerol plus fumarate. Expression of these two small polypeptides is necessary for the two large subunits to associate with the membrane. The four subunits remain associated in Triton X-100 extracts of the membrane, and a holoenzyme form of fumarate reductase containing one copy of each of the four polypeptides has been isolated. Unlike the well-characterized two-subunit form, the holoenzyme is not dependent on anions for activity and is not labile at alkaline pH. In these respects, it more closely resembles the membrane-bound activity.     

Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli

Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound four-subunit respiratory protein that shares many physical and catalytic properties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referred to as Complex II. The E. coli QFR has been overexpressed using plasmid vectors so that more than 50% of the cytoplasmic membrane fraction is composed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell proteases and oxidation factors were determined for the strains harboring the recombinant plasmid. The enzyme is extracted from the enriched membrane fraction using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange, perfusion, and gel filtration chromatography. The purified enzyme is highly active and contains all types of redox cofactors expected to be associated with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain the complete four-subunit QFR complex, diffract to 3.3 A resolution, and were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a = 96.6 A, b = 138.1 A, and c = 275.3 A. The purification and crystallization procedures are highly reproducible and the general procedure may prove useful for Complex IIs from other sources.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

In vivo detection of a three iron cluster in fumarate reductase from Escherichia coli

Escherichia coli with plasmid amplified expression of fumarate reductase was grown anaerobically on a medium containing fumarate and glycerol and investigated by electron paramagnetic resonance spectroscopy. Anaerobically harvested cells exhibit an EPR signal characteristic of a reduced [2Fe-2S] cluster. Anaerobic addition of fumarate results in diminution of the reduced [2Fe-2S] signal and the appearance of the EPR signal associated with the oxidized 3Fe cluster. The results provide the first evidence for a trinuclear iron-sulfur cluster that exists in vivo, and suggest that the 3Fe cluster in purified fumarate reductase samples is not an artifact of the isolation procedure. The significance of this observation is discussed in relation to the physiological relevance of trinuclear iron-sulfur clusters.     

Molybdenum effector of fumarate reductase repression and nitrate reductase induction in Escherichia coli.

In Escherichia coli the presence of nitrate prevents the utilization of fumarate as an anaerobic electron acceptor. The induction of the narC operon encoding the nitrate reductase is coupled to the repression of the frd operon encoding the fumarate reductase. This coupling is mediated by nitrate as an effector and the narL product as the regulatory protein (S. Iuchi and E. C. C. Lin, Proc. Natl. Acad. Sci. USA 84:3901-3905, 1987). The protein-ligand complex appears to control narC positively but frd negatively. In the present study we found that a molybdenum coeffector acted synergistically with nitrate in the regulation of frd and narC. In chlD mutants believed to be impaired in molybdate transport (or processing), full repression of phi(frd-lac) and full induction of phi(narC-lac) by nitrate did not occur unless the growth medium was directly supplemented with molybdate (1 microM). This requirement was not clearly manifested in wild-type cells, apparently because it was met by the trace quantities of molybdate present as a contaminant in the mineral medium. In chlB mutants, which are known to accumulate the Mo cofactor because of its failure to be inserted as a prosthetic group into proteins such as nitrate reductase, nitrate repression of frd and induction of narC were also intensified by molybdate supplementation. In this case a deficiency of the molybdenum coeffector might have resulted from enhanced feedback inhibition of molybdate transport (or processing) by the elevated level of the unutilized Mo cofactor. In addition, mutations in chlE, which are known to block the synthesis of the organic moiety of the Mo cofactor, lowered the threshold concentration of nitrate (< 1 micromole) necessary for frd repression and narC induction. These changes could be explained simply by the higher intracellular nitrate attainable in cells lacking the ability to destroy the effector.     

Improved conversion of fumarate to succinate by Escherichia coli strains amplified for fumarate reductase

Two recombinant plasmid Escherichia coli strains containing amplified fumarate reductase activity converted fumarate to succinate at significantly higher rates and yields than a wild-type E. coli strain. Glucose was required for the conversion of fumarate to succinate, and in the absence of glucose or in cultures with a low cell density, malate accumulated. Two-dimensional gel electrophoretic analysis of proteins from the recombinant DNA and wild-type strains showed that increased quantities of both large and small fumarate reductase subunits were expressed in the recombinant DNA strains.