title: The mechanism of high Mr thioredoxin reductase from Drosophila melanogaster.
abstract: Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57-Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489'-Cys490' in the other subunit) that transfer electrons from Cys57-Cys62 to the substrate thioredoxin. Cys489'-Cys490' functions similarly to Cys495-Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH--NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490' in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
authors.forename: Holger
authors.initials: H
authors.lastname: Bauer
authors.forename: Vincent
authors.initials: V
authors.lastname: Massey
authors.forename: L David
authors.initials: LD
authors.lastname: Arscott
authors.forename: R Heiner
authors.initials: RH
authors.lastname: Schirmer
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Charles H
authors.initials: CH
authors.lastname: Williams
mesh_terms: D000818:Animals; D002384:Catalysis; D002614:Chelating Agents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004331:Drosophila melanogaster / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D005292:Ferricyanides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D008956:Models, Chemical; D009154:Mutation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010963:Plasmodium falciparum / Q000201:enzymology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D004220:Disulfides; D005292:Ferricyanides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C007931:hexacyanoferrate III; D013879:Thioredoxins; D009249:NADP; D004492:Edetic Acid; C516124:TXNRD1 protein, human; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine; D010100:Oxygen