PDBsum entry 2bs3

Oxidoreductase

PDB id: 2bs3

Contents

Protein chains

656 a.a.

239 a.a.

255 a.a.

Ligands

FAD ×2

CIT ×2

FES ×2

F3S ×2

SF4 ×2

HEM ×4

LMT ×2

Metals

_NA ×2

Waters ×991

References listed in PDB file

Key reference

Title

Experimental support for the "e pathway hypothesis" of coupled transmembrane e- And h+ transfer in dihemic quinol:fumarate reductase.

Authors

C.R.Lancaster, U.S.Sauer, R.Gross, A.H.Haas, J.Graf, H.Schwalbe, W.Mäntele, J.Simon, M.G.Madej.

Ref.

Proc Natl Acad Sci U S A, 2005, 102, 18860-18865. [DOI no: 10.1073/pnas.0509711102]

PubMed id

16380425

Abstract

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display approximately 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 --> Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.

Figure 1.
Fig. 1. Electron and proton transfer in fumarate respiration (a) and W. succinogenes QFR (b and c). Positive and negative sides of the membrane are the periplasm and the cytoplasm, respectively. Figs. 1, 2, and 3 were prepared with a version of MOLSCRIPT (39) modified for color ramping (40) and map drawing (41) capabilities. (a) The key enzymes involved in fumarate respiration are indicated. (b) Hypothetical transmembrane electrochemical potential generation as suggested by the essential role of Glu-C66 for menaquinol oxidation by W. succinogenes QFR (16). The prosthetic groups of the W. succinogenes QFR dimer are displayed (coordinate set 1QLA, ref. 14). Distances between prosthetic groups are edge-to-edge distances in Å as defined by Page et al. (42). Also indicated are the side chain of Glu-C66 and a model of menaquinol (MKH[2]) binding. The position of bound fumarate (Fum) is taken from PDB ID code 1QLB [PDB] (14). (c) Hypothetical cotransfer of one H+ per electron across the membrane (E pathway hypothesis). The two protons that are liberated upon oxidation of menaquinol (MKH[2]) are released to the periplasm (bottom) via the residue Glu-C66. In compensation, coupled to electron transfer via the two heme groups, protons are transferred from the periplasm via the ring C propionate of the distal heme b[D] and the residue Glu-C180 to the cytoplasm (top), where they replace those protons that are bound during fumarate reduction. In the oxidized state of the enzyme, the E pathway is blocked.

Figure 4.
Fig. 4. The coupling of electron and proton flow in anaerobic (a and b) and aerobic SQRs respiration (c and d), respectively. Positive and negative sides of the membrane are described for Fig. 1. (a and b) Electroneutral reactions as catalyzed by diheme-containing QFR from W. succinogenes (a) and the hemeless QFR from E. coli. (b) MK and MKH[2], menaquinone and menaquinol, respectively. (c) Utilization of a transmembrane electrochemical potential as possibly catalyzed by diheme-containing SQR enzymes. (d) Electroneutral reaction as catalyzed by monoheme-containing SQR enzymes (complex II). Q and QH[2], ubiquinone and ubiquinol, respectively.

Secondary reference #1

Title

Wolinella succinogenes quinol:fumarate reductase and its comparison to e. Coli succinate:quinone reductase.

Author

C.R.Lancaster.

Ref.

Febs Lett, 2003, 555, 21-28.

PubMed id

14630313

Note: In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above have been manually determined.

Secondary reference #2

Title

Wolinella succinogenes quinol:fumarate reductase-2.2-A resolution crystal structure and the e-Pathway hypothesis of coupled transmembrane proton and electron transfer.

Author

C.R.Lancaster.

Ref.

Biochim Biophys Acta, 2002, 1565, 215-231. [DOI no: 10.1016/S0005-2736(02)00571-0]

PubMed id

12409197

Secondary reference #3

Title

A third crystal form of wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction.

Authors

C.R.Lancaster, R.Gross, J.Simon.

Ref.

Eur J Biochem, 2001, 268, 1820-1827. [DOI no: 10.1046/j.1432-1327.2001.02053.x]

PubMed id

11248702

Figure 2.
Fig. 2. The site of fumarate reduction in subunit A (stereo views). The QFR crystal form ‘C’ Arg A301 carbon atoms are drawn in pink and its nitrogen atoms in light blue. (A) Electron density maps from the refined model of crystal form ‘C’ at 3.1 Å resolution. Contour levels are 1.0 (2F[o] - F[c,] blue) and 3.0 (F[o] - F[c,] green, with the malonate molecule omitted from the phase calculation). Due to insufficient density in the 2F[o] - F[c] map, the side chain of Arg A301 has been assigned zero occupancy. See text for details. (B) Comparison of W. succinogenes QFR crystal forms ‘C’ (PDB entry 1E7P, carbon atoms in yellow, complex with malonate) and ‘B’ (PDB entry 1QLB, carbon atoms in green, complex with fumarate). The isolated red spheres correspond to the oxygen atoms of two water molecules in PDB entry 1QLB. (C) Comparison of the crystal structures of the fumarate reducing sites in W. succinogenes QFR crystal form ‘C’ (PDB entry 1E7P, carbon atoms in yellow), in E. coli QFR (PDB entry 1FUM, carbon atoms in white), and in the S. frigidimarina soluble flavocytochrome c[3] (PDB entry 1QJD, carbon atoms in grey). The different dicarboxylate compounds included in the models are malonate (1E7P), oxaloacetate (1FUM), and a malate-like intermediate (1QJD). Residues are numbered according to W. succinogenes QFR.

Figure 3.
Fig. 3. Possible mechanism of fumarate reduction in W. succinogenes QFR involving the residues shown in Fig. 2 Go-. Hydride transfer from the N5 of FAD to the -methenyl of fumarate (in blue) is coupled to proton transfer to the position of the substrate from the side chain of Arg A301.

The above figures are reproduced from the cited reference with permission from the Federation of European Biochemical Societies

Secondary reference #4

Title

Essential role of glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by wolinella succinogenes fumarate reductase.

Authors

C.R.Lancaster, R.Gorss, A.Haas, M.Ritter, W.Mäntele, J.Simon, A.Kröger.

Ref.

Proc Natl Acad Sci U S A, 2000, 97, 13051-13056. [DOI no: 10.1073/pnas.220425797]

PubMed id

11186225

Figure 1.
Fig. 1. A working hypothesis. Two distal cavities (purple) in subunit C of the original structure of W. succinogenes QFR (PDB entry 1QLA) as detected with the program VOIDOO (33) and a working model of menaquinol binding (green) are shown. To accommodate the quinol head group in its current tentative position between the cavities, amino acid side-chain movements from their original positions (blue) to positions drawn in red are required as derived from energy minimization simulations with CNS. The heme group shown is the distal heme b[D]. In this orientation, the periplasm is at the bottom and the rest of the QFR complex extends beyond the top and the right of the figure. Figs. 1, 2, and 4 were prepared with a version of MOLSCRIPT (34) modified for color ramping (35) and map drawing (36) capabilities.

Figure 4.
Fig. 4. Transmembrane electrochemical potential generation by W. succinogenes QFR coupling the two-electron oxidation of menaquinol (MKH[2]) to menaquinone (MK) to the two-electron reduction of fumarate to succinate. The positive (+) and negative ( ) sides of the membrane are indicated. The prosthetic groups of the QFR dimer are displayed (coordinate set PDB entry 1QLA; ref. 5). Distances between prosthetic groups are edge-to-edge distances in Å as defined in ref. 37. Distances shorter than 14 Å (i.e., within one QFR monomer, but not between the two monomers of the dimer) are considered to be relevant for physiological electron transfer. Also drawn are the side chains of Glu-C66 (in red) and of the subunit C Trp residues (purple). The latter are markers for the hydrophobic surface-to-polar transition zone of the membrane. The position of bound fumarate (Fum) is taken from PDB entry 1QLB (5). The tentative model of menaquinol binding (drawn in green) is taken from Fig. 1. Its edge-to-edge distance to heme b[D] is 6.7 Å.

Secondary reference #5

Title

Structure of fumarate reductase from wolinella succinogenes at 2.2 a resolution.

Authors

C.R.Lancaster, A.Kröger, M.Auer, H.Michel.

Ref.

Nature, 1999, 402, 377-385. [DOI no: 10.1038/46483]

PubMed id

10586875

Figure 1.
Figure 1 Representative parts of the experimental electron-density maps for crystal form A calculated with the MIRAS phases after density modification and phase extension to 2.2 ? resolution. C, N, O, P and S atoms are shown in grey, blue, red, light green and green, respectively; haem iron centres are shown in orange. Contour levels are 1.0 (green) and 9.0 (red) above the mean density of the map. Figs 1-4 and 6 were prepared with a version of Molscript46 modified by R. Esnouf for colour ramping47 and map drawing48 capabilities. a, b, The two haem b molecules (b[P] in the top half; b[D] in the bottom half of each panel) and the side chains of some neighbouring residues in the transmembrane region. c, The covalently bound FAD prosthetic group.

Figure 2.
Figure 2 The three-dimensional structure of fumarate reductase. a, The fumarate reductase dimer viewed parallel to the membrane. The polypeptide backbones of the two A subunits are shown in blue and light blue, those of the two B subunits in red and pink, and those of the C subunits in green and yellow. Subunit A contains a covalently bound FAD. Subunit B contains three iron-sulphur clusters (Fe[2]S[2], Fe[4]S[4] and Fe[3]S[4]). The membrane-embedded subunit C contains two haem b molecules. b, View of the transmembrane helices of the subunit C dimer along the membrane normal from the cytoplasmic side. One monomer is colour-coded from blue (N terminus) to yellow (C terminus), the other from yellow (N terminus) to red (C terminus)). The transmembrane helices are labelled I, II, IV, V and VI (ref. 22). Secondary structures were assigned using DSSP49. Figure rendered with Raster3D^50.

The above figures are reproduced from the cited reference with permission from Macmillan Publishers Ltd