Acid−Base Catalysis in the Mechanism of Thioredoxin Reductase from Drosophila melanogaster^†

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Like other members of the pyridine nucleotide-disulfide oxidoreductase enzyme family, the enzyme from Drosophila melanogaster is a homodimer, and each catalytically active unit consists of three redox centers:  FAD and an N-terminal Cys-57/Cys-62 redox-active disulfide from one monomer and a Cys-489‘/Cys-490‘ C-terminal redox-active disulfide from the second monomer. Because dipteran insects such as D. melanogaster lack glutathione reductase, thioredoxin reductase (DmTrxR) is particularly important; in addition to its normal functions, it also reduces GSSG for antioxidant protection. DmTrxR, used as a model for the enzyme from the malaria vector, Anopheles gambiae, has been shown to cycle in catalysis between the two-electron and four-electron reduced states, EH₂ and EH₄ [Bauer, H. et al. (2003) J. Biol. Chem. 278, 33020−33028]. His-464‘ acts as an acid−base catalyst of the dithiol−disulfide interchange reactions required in catalysis. The H464‘Q enzyme has only 2% of the wild-type activity, emphasizing the importance of this residue. The pH dependence of V_max for wild-type DmTrxR has pK_a values of 6.4 and 9.3 on the DmTrxR−DmTrx-2 complex, whereas H464‘Q DmTrxR only has an observable pK_a at 6.4, indicating that the pK_a at pH 9.3 is contributed mainly by His-464‘. The pK_a at pH 6.4 has been assigned to Cys-57 and Cys-490‘; the thiolate on Cys-490‘ is the nucleophile in the reduction of Trx. In contrast to wild-type DmTrxR, H464‘Q DmTrxR does not stabilize a thiolate−FAD charge-transfer complex in the presence of excess NADPH. The rates of steps in both the reductive and the oxidative half-reactions are markedly diminished in H464‘Q DmTrxR as compared to those of wild-type enzyme, indicating that His-464‘ is involved in both half-reactions.