Structural insights into the peroxidase activity and inactivation of human peroxiredoxin 4

Abstract

Prx4 (peroxiredoxin 4) is the only peroxiredoxin located in the ER (endoplasmic reticulum) and a proposed scavenger for H2O2. In the present study, we solved crystal structures of human Prx4 in three different redox forms and characterized the reaction features of Prx4 with H2O2. Prx4 exhibits a toroid-shaped decamer constructed of five catalytic dimers. Structural analysis revealed conformational changes around helix α2 and the C-terminal reigon with a YF (Tyr-Phe) motif from the partner subunit, which are required for interchain disulfide formation between Cys87 and Cys208, a critical step of the catalysis. The structural explanation for the restricting role of the YF motif on the active site dynamics is provided in detail. Prx4 has a high reactivity with H2O2, but is susceptible to overoxidation and consequent inactivation by H2O2. Either deletion of the YF motif or dissociation into dimers decreased the susceptibility of Prx4 to overoxidation by increasing the flexibility of Cys87.

Glutathione Peroxidase 7 Utilizes Hydrogen Peroxide Generated by Ero1α to Promote Oxidative Protein Folding

“…The two-cysteine Prx4 was reported to catalyze disulfide formation in vitro and in vivo (28,39). However, Prx4 can be inactivated by H 2 O 2 , and no enzymes that are capable of reducing over-oxidized Prxs (such as sulfiredoxin) have been found so far in the ER (36). Different from Prx4, GPx7 is hardly inactivated at a sub-millimolar concentration of H 2 O 2 (unpublished data), implying that GPx7 is either resistant to overoxidation or protected by PDI.…”

“…over-oxidized and inactivated by H 2 O 2 (36), and Prx4 knockout mice have only a minor phenotype (14), implying the existence of other H 2 O 2 scavengers in the secretory compartment. Recently, two ER-located glutathione peroxidases (GPx7 and GPx8) were reported to catalyze protein refolding in the presence of PDI and H 2 O 2 in vitro, with GPx7 being more efficient (22).…”

Transit of H2O2 across the endoplasmic reticulum membrane is not sluggish

“…By contrast, depletion of the ER-luminal high-abundance-high-affinity-high-turnover-peroxidase peroxiredoxin 4 [40,41] does not cause similar leakage of Ero1 -derived H 2 O 2 into the cytosol [39]. Thus, the shielding of the cytosol against Ero1 -derived H 2 O 2 takes place at the Ero1 -GPx8 interface through catalytic elimination [42].…”

Peroxiredoxin 4 Improves Insulin Biosynthesis and Glucose-induced Insulin Secretion in Insulin-secreting INS-1E Cells

“…This catalytic reaction can be accomplished by three recently described ER-localized peroxidases, Prdx4, GPx7, and GPx8 (16,19,20). Among them, Prdx4 is ubiquitously expressed and enriched in highly active secretory tissues (37 (27), indicating that H 2 O 2 preferentially reacts with the peroxidatic cysteine within Prdx4 rather than with cysteines in GPx7, GPx8, and PDI or other ER proteins (16,35). The essential role of disulfide bond formation in assembly and processing of insulin (38) and the ability of Prdx4 to couple H 2 O 2 metabolism with disulfide bond formation (19,20) motivated us to characterize the functional significance of the ER-specific Prdx4 on ␤-cell function with emphasis on insulin content and secretion during stimulation with nutrient secretagogues.…”