Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II, and the identity of the ligand in the "oxaloacetate-inhibited" state.
Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115-1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the "malate-like intermediate" found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108-1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1-9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.
Huang
Li-Shar
LS
Physical Biosciences Division, Lawrence Berkeley National Lab., MS 64-0121, 1 Cyclotron Road, Berkeley CA 94720, USA.
Shen
John T
JT
Wang
Andy C
AC
Berry
Edward A
EA
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D042963:Electron Transport Complex II / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008024:Ligands; D008293:Malates / Q000378:metabolism; D008314:Malonates / Q000378:metabolism; D062907:Oxaloacetic Acid / Q000378:metabolism / Q000494:pharmacology*; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D005650:Fumarates; D007506:Iron-Sulfur Proteins; D008024:Ligands; D008293:Malates; D008314:Malonates; D062907:Oxaloacetic Acid; C030298:malic acid; C032005:fumaric acid; D042963:Electron Transport Complex II