Four Cys residues in heterodimeric 2-oxoacid:ferredoxin oxidoreductase are required for CoA-dependent oxidative decarboxylation but not for a non-oxidative decarboxylation.
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 320nm 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.
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Zhen
Z
Yan
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Shinya
S
Fushinobu
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan. Electronic address: atwakag@mail.ecc.u-tokyo.ac.jp.
Takayoshi
T
Wakagi
D000105:Acetyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D003653:Decarboxylation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005609:Free Radicals; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
D019843:Archaeal Proteins; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D013455:Sulfur; D000105:Acetyl Coenzyme A; D007501:Iron; D007658:Ketone Oxidoreductases; C023096:2-oxoglutarate synthase; D003545:Cysteine; D000409:Alanine
Iron–sulfur cluster; Non-oxidative decarboxylation; Oxidative decarboxylation; Sulfolobus tokodaii