Abstract
Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.
Highlights
Peroxiredoxin IV metabolizes endoplasmic reticulum-derived hydrogen peroxide
We show that Peroxiredoxin IV (PrxIV) has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues
We have previously demonstrated that the purified recombinant protein is disulfide-linked between the peroxidatic and resolving cysteine residues [17], so we surmised that crystals formed would be of the oxidized disulfide-bonded form and that crystals formed in the presence of DTT would be of the reduced form
Summary
Peroxiredoxin IV metabolizes endoplasmic reticulum-derived hydrogen peroxide. Results: Peroxiredoxin IV structures reveal an unusually stable decamer and a sulfenylated intermediate in the enzymatic cycle. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. The peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue. Preventing the formation of the interchain disulfide between the peroxidatic and resolving cysteine by mutation of the resolving cysteine to alanine resulted in a mutant that was insensitive to hyper-oxidation of the peroxidatic cysteine Structural analysis of this mutant shows that the enzyme can adopt the LU conformation when the peroxidatic cysteine is sulfenylated. This novel peroxiredoxin structure reveals a potential intermediate in the enzymatic cycling and supports the idea that sulfenylation of the peroxidatic cysteine causes the conformational shift from the FF to the LU form and prevents further oxidation occurring
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