PDBsum entry 1qjd

Oxidoreductase

PDB id: 1qjd

Contents

Protein chain

568 a.a. *

Ligands

HEC ×4

FAD

TEO

GOL

Metals

_NA

Waters ×576

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Structural and mechanistic mapping of a unique fumarate reductase.

Authors

P.Taylor, S.L.Pealing, G.A.Reid, S.K.Chapman, M.D.Walkinshaw.

Ref.

Nat Struct Biol, 1999, 6, 1108-1112. [DOI no: 10.1038/70045]

PubMed id

10581550

Abstract

The 1.8 A resolution crystal structure of the tetraheme flavocytochrome c3, Fcc3, provides the first mechanistic insight into respiratory fumarate reductases or succinate dehydrogenases. The multi-redox center, three-domain protein shows a 40 A long 'molecular wire' allowing rapid conduction of electrons through a new type of cytochrome domain onto the active site flavin, driving the reduction of fumarate to succinate. In this structure a malate-like molecule is trapped in the enzyme active site. The interactions between this molecule and the enzyme suggest a clear mechanism for fumarate reduction in which the substrate is polarized and twisted, facilitating hydride transfer from the reduced flavin and subsequent proton transfer. The enzyme active site in the oxidized form is completely buried at the interface between the flavin-binding and the clamp domains. Movement of the cytochrome and clamp domains is postulated to allow release of the product.

Figure 3.
Figure 3. The active site of Fcc[3]. a, Stereo MOLSCRIPT^30 representation of the environment of the modified substrate. Hydrogen bonds are shown as red dashed lines. Other short contacts are shown as green dashed lines. Distances are also tabulated showing hydrogen bonds and short nonbonded contacts (<3.1 Å) involving the modified substrate. b, Connolly surfaces of the available active site volume (green) and substrate-occupied volume (red). The Connolly surface of the available volume in the substrate binding site (transparent green surface) is almost completely filled by the substrate intermediate molecule. The Connolly surface of the substrate intermediate molecule shown as a wire representation colored red. c, An overlay of a modeled planar fumarate (magenta) and the modified substrate. Short contacts between the modeled fumarate molecule and the two clamping methionine residues are shown. The resulting twist in the conformation of the carboxyl group is important in the enzyme mechanism.

Figure 4.
Figure 4. Reaction mechanisms at the Fcc[3] active site. a, Schematic representation of the mechanism of fumarate reduction by Fcc[3.] Catalysis is initiated by the binding of fumarate at the active site. The C1 carboxylate group of fumarate (to the left in this representation) is twisted out of plane by the closure of the clamp domain and the resulting steric constraints imposed by the side chains of Met 236 and Met 375 (Fig. 3d) and by hydrogen bonding to His 365. The substrate C4 carboxylate is bound in a very positively charged environment involving interactions with His 504, Arg 544 and Arg 402. The combination of these effects results in polarization of the C2−C3 bond with the build up of positive charge at C2, facilitating hydride transfer from N5 of the reduced flavin to the si-face of the substrate. Arg 402 which is only 2.99 Å from C3, is ideally positioned for proton transfer, resulting in the formation of the product, succinate. b, Proposed mechanism for the formation of the hydrated intermediate at the active site of the oxidized enzyme. In the oxidized flavocytochrome c[3], as is the case in the crystal, there is no hydride available to attack the substrate C2 atom. Instead, water acts as a nucleophile attacking the re-face. The resulting intermediate, which is shown in the electron density to have R-stereochemistry at C2, is trapped at the active site in the crystal. c, Conservation of active site residues in fumarate reductases and succinate dehydrogenases. Segments of the sequence of Fcc[3] are aligned with the corresponding regions of the flavoprotein subunits of the fumarate reductases from E. coli (frda_ecoli) and Wolinella succinogenes (frda_wolsu) and the succinate dehydrogenases from E.coli (dhsa_ecoli) and Saccharomyces cerevisiae (dhsa_yeast) to highlight the conservation around active site residues. The following residues are highlighted in red: His 365 and Thr 377 (top left), Arg 402 (top right), His 504 (bottom left) and Arg 544 (bottom right).

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (1999, 6, 1108-1112) copyright 1999.