PDBsum entry 1kek

Oxidoreductase

PDB id

1kek

Contents

			Protein chains

					1231 a.a. *

			Ligands

					SF4 ×6


					HTL ×2


					CO2 ×2

			Metals

					_MG ×2


					_CA ×2

			Waters ×1893

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase.

Authors

E.Chabrière, X.Vernède, B.Guigliarelli, M.H.Charon, E.C.Hatchikian, J.C.Fontecilla-Camps.

Ref.

Science, 2001, 294, 2559-2563. [DOI no: 10.1126/science.1066198]

PubMed id

11752578

Abstract

In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.



	Figure 3. Fig. 3. (A) Stereo pair of the acetyl-ThDP moiety and the bound CO[2 ]molecule of PFOR and their protein environment. (B) Stereo pair of the superposition of a part of the active site of PFOR in the uncomplexed (green), and radical forms. The movements of the thiazole ring and the side chains of Asn996 and Tyr994 are concerted, and the S1 atom from the thiazole ring keeps its hydrogen bond to Asn996 in the two conformations. Part (A) was prepared using Molscript (39) and Raster3d (40); (B) was prepared with Turbo-Frodo (38).		Figure 4. Fig. 4. Postulated mechanism of acetyl-CoA synthesis by PFOR. Only the thiazolium ring moiety of ThDP is fully depicted (R and R' as in Fig. 1, B and C). (A) Deprotonated carbanion species (see Fig. 1A). The proton is putatively bound to 4'-iminopyridimine (not shown). (B) Pyruvate decarboxylation and hypothetical enamine formation; the CO[2] reaction product stays in the active site. (C) One electron transfer from the active site to one of the [4Fe4S] clusters. Hypothetical n cation radical formation. (D) Observed /n cation radical with a long C2-C2 bond (27) and a bent thiazole ring (Fig. 2). Note that (i) ketonization of the enamine (B) upon radical formation (C) and (ii) tautomerization of the C5-C4 double bond to a C4-C4 double bond, in going from (B) to (C), are required to explain the observed stereochemistry of the adduct. The net result of these two rearrangements is a significant reduction in the aromaticity of the thiazole ring. Because this process is generally considered to be unfavorable, the protein environment is thought to play a key role in the stabilization of (C) and (D). The loss of one electron from the active site and the bending of the thiazole ring are shown here as a single step because we do not know the detailed sequence of events. (E) Hypothetical fragmented C-C bond resulting in carbocation and acetyl radical species (28, 29). Upon fragmentation, the aromaticity of the thiazole ring is thought to be restored (A), closing the cycle. (F) Acetyl-CoA synthesis through condensation of a thiyl CoA radical with the acetyl radical. Although the reaction is shown in the direction of acetyl-CoA synthesis, PFORs are capable of catalyzing the reverse reaction.

PROCHECK

	Headers