Four Cys residues in heterodimeric 2-oxoacid:ferredoxin oxidoreductase are required for CoA-dependent oxidative decarboxylation but not for a non-oxidative decarboxylation

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• 作者：Zhen Yan ; Shinya Fushinobu ; Takayoshi Wakagi ; ^{atwakag@mail.ecc.u-tokyo.ac.jp" class="auth_mail}
• 关键词：OFOR ; 2-oxoacid ; ferredoxin oxidoreductase ; EPR ; electron paramagnetic resonance ; ThDP ; thiamine diphosphate ; Fe&ndash ; S ; iron&ndash ; sulfur cluster ; PFOR ; pyruvate ; ferredoxin oxidoreductase ; TCA ; tricarboxylic acid
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：April, 2014
• 年：2014
• 卷：1844
• 期：4
• 页码：736-743
• 全文大小：802 K

文摘

Heterodimeric 2-oxoacid:ferredoxin oxidoreductase (OFOR) from Sulfolobus tokodaii (StOFOR) has only one [4Fe-4S]^2 + cluster, ligated by 4 Cys residues, C12, C15, C46, and C197. The enzyme has no other Cys. To elucidate the role of these Cys residues in holding of the iron-sulfur cluster in the course of oxidative decarboxylation of a 2-oxoacid, one or two of these Cys residues was/were substituted with Ala to yield C12A, C15A, C46A, C197A and C12/15A mutants. All the mutants showed the loss of iron-sulfur cluster, except the C197A one which retained some unidentified type of iron-sulfur cluster. On addition of pyruvate to OFOR, the wild type enzyme exhibited a chromophore at 320 nm and a stable large EPR signal corresponding to a hydroxyethyl-ThDP radical, while the mutant enzymes did not show formation of any radical intermediate or production of acetyl-CoA, suggesting that the intact [4Fe-4S] cluster is necessary for these processes. The stable radical intermediate in wild type OFOR was rapidly decomposed upon addition of CoA in the absence of an electron acceptor. Non-oxidative decarboxylation of pyruvate, yielding acetaldehyde, has been reported to require CoA for other OFORs, but StOFOR catalyzed acetaldehyde production from pyruvate independent of CoA, regardless of whether the iron-sulfur cluster is intact [4Fe-4S] type or not. A comprehensive reaction scheme for StOFOR with a single cluster was proposed.