The dicarboxylate site, represented by a green space-filling model of the “malate-like ligand” found in the oxaloacetate-inhibited complex, is in the flavoprotein subunit between the CAP domain (magenta) and the rest of the flavoprotein (yellow). The region around the dicarboxylate site is expanded in the lower panel, and key residues involved in binding TEO are labeled. Returning to the overall structure (upper panel), a series of three iron-sulfur clusters transfers electrons between Flavin (blue) at the dicarboxylate site and the quinone binding site (brown ubiquinone headgroup, labeled). The coordinates are from structure 2H88, except for ubiquinone, which was modeled in for the purpose of this illustration based on structures 1NEK (E. coli Complex II) and on the binding of carboxin in structure 2FBW.

Note how the C1 carboxylate is twisted out of the plane of the rest of the molecule which lies flat against the FAD isoalloxazine ring. Upper panel: The coordinates from structure 2H88 (monomer 1) are shown together with a 2Fo-Fc map from that structure contoured at 1.7 σ. The view is from the direction of His 364 which has been removed, together with its H-bond to the C2 oxygen. Arg 297 and Arg408 are labeled. His253 can be seen H-bonding TEO from the top. To the left are the backbone N of Glu266 (upper left), O_γ of Thr265, and the backbone N of Gly62. One more H-bond from the backbone N of Ala411 to the carboxylate on the right is not shown. Lower panel: The coordinates of 2H88 (elemental colors) are superimposed on the same region in the Shewanella FCc structure 1QJD (pastel blue) based on aligning 59 Cα atoms in 8 stretches of the FAD and CAP domains of the flavoproteins (rmsd 0.39 Å). Note the general similarity and the close superpositioning of the ligands.

(A)The spectral effect of OAA binding was determined by following the dissociation of OAA from Complex II in the presence of OAA transaminase scavaging system [5]. Complex II was diluted to 1 ml in buffer A supplemented with 10 mM glutamate, and two initial spectra were recorded. Then 1 μl of transaminase was added and scans were recorded repeatedly during the stripping process. The average of the spectra of Complex II before adding transaminase was subtracted from all, and spectra before and at 0, 6.8, 28.6, 43.4, and 66 min after the addition are plotted. Then (not shown) dithionite was added to obtain the fully reduced spectrum from which the enzyme concentration was determined. (B). Time course of dissociation of OAA. A spectrum representative of the OAA-induced change was obtained form the spectra of figure 4a. The experiment of (A) was repeated several times, and one of these experiments, in which the decay was slightly faster, was selected for determining the spectrum of the fully-stripped enzyme. The spectra were fit to a linear combination of spectra of the as-purified Complex II and the OAA-induced change. The resulting amplitude of the OAA effect was plotted against time and fit with a single exponential. The t_∞ value obtained from the fit was taken as the amount of the OAA-effect spectrum to subtract from the as-purified spectrum to obtain the spectrum of the fully-stripped enzyme, which was scaled to 1 μM based on the concentration determined from the dithionite-reduced spectrum above. The spectrum of the OAA effect was scaled by assuming the site was fully occupied with OAA in the malate-treated enzyme. The spectra were then re-fit using this normalized OAA effect spectrum and the stripped Complex II spectrum, to obtain the data shown, in which the OAA effect decays toward zero. The resulting value for Complex II concentration was constant at 5.7 μM, and the OAA effect before adding transaminase was 2.4 μM. Inset: The experiment was repeated at different temperatures and the log of the first-order rate constant was plotted against reciprocal temperature to obtain an approximate activation energy of 25.4 kcal/mol (106 kJ/mol).

The blue trace is an experimental spectrum of the Complex II preparation from which the crystal leading to the 2H88 structure was grown. It had been treated with malonate at room temperature for 3 hours and sedimented through a gradient containing 5 mM fumarate as described in materials and methods. The green trace is the best fit to the experimental trace using a linear combination of the spectra of Complex II, the fumarate effect, and the OAA effect. The required magnitude of each of these is shown (respectively light blue, magenta, and brown).

The 2Fo-Fc map is contoured at 15 σ (magenta) to show the iron atoms, and at 6 σ (cyan) to show the sulfurs as well. The irons and sulfurs, separated by about 2.2 Å, are well resolved in this structure.

Stereo view of malonate in the dicarboxylate site of complex II. The protein was crystallized in the presence of malonate. as described in Materials and Methods. Coordinates and 2Fo-Fc map from the structure deposited as 2H89. The view is nearly the same as in Figure 3.

Difference spectra representing the effects of various ligands on the spectrum of fully oxidized Complex II. Preliminary standard spectra for the spectral effects of OAA, malonate, fumarate, and 3-nitropropioninc acid were determined as described in the text. The magnitude is on an arbitrary scale.

About 5 mM Complex II in buffer a was monitored spectrally before and after addition of 10 mM sodium fumarate. The temperature was 20°C. (A). Time course: Each time point represents one spectrum. Fumarate was added after the second spectrum (arrows). Each spectrum was fit to a linear combination of the empty-site complex, the OAA effect, and the fumarate effect. The amount of each spectrum required to reconstitute the experimental spectrum is plotted against time. Panels B and C show (blue traces) difference spectra: 14 min vs before fumarate (B) and 460 minutes vs 14 min (C). The green curves show the fit to these two spectra and the red and magenta curves show the amount of the OAA and fumarate spectral effects required to fit them. The slow phase (C) consists mainly of disappearance of the fumarate effect and appearance of the OAA effect, suggesting that fumarate is being converted into the tightly-bound ligand of the OAA-inhibited complex.