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Structure of the Escherichia coli Fumarate Reductase Respiratory Complex

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Structure of the Escherichiacoli Fumarate ReductaseRespiratory ComplexTina M. Iverson,1Ce´sar Luna-Chavez,2Gary Cecchini,2*Douglas C. Rees3*The integral membrane protein fumarate reductase catalyzes the final step ofanaerobic respiration when fumarate is the terminal electron acceptor. Thehomologous enzyme succinate dehydrogenase also plays a prominent role incellular energetics as a member of the Krebs cycle and as complex II of theaerobic respiratory chain. Fumarate reductase consists of four subunits thatcontain a covalently linked flavin adenine dinucleotide, three different iron-sulfur clusters, and at least two quinones. The crystal structure of intactfumarate reductase has been solved at 3.3 angstrom resolution and demon-strates that the cofactors are arranged in a nearly linear manner from themembrane-bound quinone to the active site flavin. Although fumarate reduc-tase is not associated with any proton-pumping function, the two quinones arepositioned on opposite sides of the membrane in an arrangement similar to thatof the Q-cycle organization observed for cytochrome bc1.Because oxygen has a high affinity forelectrons, aerobic respiration represents avery favorable form of energy metabolism.However, in the absence of oxygen, manymicroorganisms can obtain energy throughanaerobic respiratory processes that resultin the reduction of alternate terminal accep-tors (1). One of the most widespread accep-tors is fumarate (2), which is reduced tosuccinate by fumarate reductase, an integralmembrane protein containing flavin ade-nine dinucleotide (FAD) and iron-sulfurclusters (3). The electron donor for thisreaction is reduced menaquinone, whichcommonly serves as a membrane-soluble,mobile electron carrier between respiratorycomplexes. The most extensively charac-terized fumarate reductase, from Esche-richia coli, has a total molecular mass of121 kD in four subunits. It consists of twowater-soluble subunits, the flavoprotein (66kD) and iron-sulfur protein (27 kD) sub-units, and two membrane anchor subunits(15 and 13 kD), which are the products ofthe frdABCD genes, respectively (4 ). Theflavoprotein (Fp) subunit contains the cat-alytic site for fumarate reduction and suc-cinate oxidation at a covalently linked FAD(5 ), while the iron-sulfur protein subunit(Ip) contains three different types of iron-sulfur clusters, [2Fe:2S], [4Fe:4S], and[3Fe:4S], which have been spectroscopical-ly characterized (6 ). At least two sites as-sociated with the membrane anchor sub-units have been proposed to bind the qui-nones that are involved in electron transferreactions of the enzyme (7 ).Fumarate reductase catalyzes the re-verse reaction of succinate dehydrogenase,which participates in both the aerobic res-piratory chain as complex II and in theKrebs cycle (3). These two proteins exhibitsubstantial similarities in amino acid se-quence, cofactor composition, and mecha-nism. Indeed, under certain conditions, oneenzyme can functionally replace the otherand support bacterial growth (8). Becauseof the central role of fumarate reductaseand succinate dehydrogenase in respiration,mutations in these complexes can have sub-stantial metabolic consequences. In bacte-ria, mutations in fumarate reductase cansignificantly retard growth under appropri-ate conditions (9). In higher organisms,mutations of succinate dehydrogenase havebeen linked to oxidative stress and aging innematodes (10) and to Leigh’s syndrome inhumans (11). Historically, succinate dehy-drogenase was one of the most widely stud-ied enzymes during the development ofenzymology. Early studies resulted in thediscoveries of nonheme iron and covalentlybound flavin in proteins (12). To provide aframework for addressing the functionalproperties of fumarate reductase and succi-nate dehydrogenase, we have solved thestructure of the E. coli fumarate reductaseat 3.3 Å resolution. Here we describe thefold of the polypeptides and location of thecofactors, and the functional implicationsof this structural arrangement.Structure Determination andOverall FoldFumarate reductase from E. coli was purifiedand crystallized in the presence of the non-ionic detergent Thesit (13). The structure wassolved by multiple wavelength anomalousdiffraction (MAD), with data collected atthree wavelengths near the Fe K edge (14 )(Table 1). The iron-sulfur clusters and trans-membrane helices were striking in the initialmaps calculated at 4 Å resolution, and thestructure was solved by iterative combinationof density modification, noncrystallographicsymmetry averaging, model building, and re-finement (15). The final model has been re-fined to values of Rcrystof 22.2% and Rfreeof29.2% at 3.3 Å resolution with reasonablestereochemistry (15, 16).The four subunits in fumarate reductaseare arranged in a complex resembling theletter “q,” with the top of the “q” generatedby the Fp and Ip subunits (diameter ⬃70 Å),while the tail of the “q” (length 110 Å)contains the membrane anchor subunits (Fig.1, A and B). The orientation of fumaratereductase in the cell membrane is such thatthe Fp and Ip subunits are located in thecytoplasm (equivalent to the mitochrondrialmatrix for succinate dehydrogenase). Inthese crystals, two fumarate reductase com-plexes, which are related by a twofold axisapproximately parallel to the membranenormal, are present per asymmetric unit.These two complexes associate throughtheir transmembrane regions. Contacts withneighboring molecules related by crystallo-graphic symmetry also occur in the mem-brane-spanning region, creating a continu-ous membrane-spanning region throughoutthe crystal (Fig. 1C). Despite the sugges-tiveness of this arrangement, there is noevidence that a dimer is physiologicallyrelevant, unlike the situation with cyto-chrome bc1(17 ). Additionally, the contactregion between fumarate reductase mole-cules in the crystals is relatively small(⬃325 Å2)(18) and is unlikely to supportformation of a stable dimer (Fig. 1B).The Fp (FrdA) subunit is organizedaround an FAD/NAD(P) (nicotinamide ade-nine dinucleotide phosphate) binding domainformed by residues A1 to A50, A130 toA231, and A354 to A414 (Fig. 2A). Thisdomain structure includes a Rossmann-typefold that provides the binding site for FAD.The FAD is further associated with the fla-voprotein through a covalent bond betweenthe flavin C8A methyl group and the N␧ atomof the side chain of His A44. The remainingresidues of this subunit, A51 to A129, A232to A353, and A415 to A575, are


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