Structural snapshots of yeast alkyl hydroperoxide reductase Ahp1 peroxiredoxin reveal a novel two-cysteine mechanism of electron transfer to eliminate reactive oxygen species

Abstract

Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C(P)) to Cys-62 and the resolving cysteine (C(R)) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ∼2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 Å resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C(P) and C(R). An intermolecular C(P)-C(R) disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.

FIGURE 1.

Assignment of the C_P and C_R of Ahp1. A, shown are peroxidase activity assays. Assays were performed with Ahp1 (black inverted triangles), Ahp1C31S (red triangles), Ahp1C62S (blue circles), and Ahp1C120S (dark cyan diamonds). B, shown is multiple-sequence alignment of Ahp1 against other Prx5 subfamily proteins from different organisms. Both Cys-62 and Cys-31 of Ahp1 are conserved through homologs from several species of fungi and bacteria. In contrast, the homologs from several species of higher eukaryotes lack the corresponding resolving Cys-31 (C_R) and have a conserved resolving C_R at the C-terminal region. The multi-sequence alignment was performed using programs MultAlin (52) and ESPrint (53). The secondary structural elements of reduced Ahp1 are displayed above the sequences. The residues contributed to dimer interface were indicated with black (hydrogen bonds) and blue (hydrophobic interactions) triangles. Cys-120 was indicated with a red dot. All sequences were downloaded from the NCBI data base (www.ncbi.nlm.nih.gov). The sequences are (NCBI accession numbers codes are in parentheses) S. cerevisiae Ahp1 (NP_013210.1), Vanderwaltozyma polyspora Kpol_1004p59 (XP_001645540.1), Zygosaccharomyces rouxii ZYRO0D06248p (XP_002496705.1), Debaryomyces hansenii DEHA2G17864p (XP_462316.1), Aspergillus oryzae pmp20 (XP_001727651.1), Schizophyllum commune SCHCODRAFT_67750 (XP_003031587.1), Polynucleobacter necessarius redoxin domain-containing protein (YP_001154958.1), Ralstonia solanacearum peroxiredoxin (YP_003744570.1), Methylococcus capsulatus anti-oxidant AhpC-TSA family protein (YP_112582.1), Burkholderia vietnamiensis redoxin domain-containing protein (YP_001118388.1), Janthinobacterium Marseille peroxiredoxin (YP_001354699.1), Arabidopsis thaliana Prx-2D (NP_564763.1), Drosophila melanogaster peroxiredoxin 5 (NP_001027191.1), Danio rerio Prx 5 (NP_001019577.1), Xenopus laevis Prx 5 (NP_001085580.1), Mus musculus Prx 5 (NP_036151.1), and Homo sapiens Prx 5 (NP_036226.1).

FIGURE 2.

The reaction kinetics of Ahp1 toward the substrates. A, t-BOOH. B, Trx2. The plots were fitted by using the Hill equation.

FIGURE 3.

Structures of the oxidized and reduced Ahp1. A, shown is a schematic of oxidized Ahp1 homodimer structure. The side chains of Cys-31, Cys-62, and Cys-120 are shown as sticks, and sulfur atoms are shown as yellow balls. Residues of subunit B are labeled with a prime. B, shown is a schematic of reduced Ahp1 homodimer structure. The side chains of Cys-31, Cys-62, and Cys-120 are shown as sticks, and sulfur atoms shown as yellow balls. C, shown is a detailed representation of the dimer interface in oxidized Ahp1. Disulfide bonds and hydrogen bonds are indicated in C, and hydrophobic interactions are indicated in D. The involved residues are shown as sticks, and sulfur atoms of cysteines are shown as yellow balls. The two subunits of Ahp1 are shown in green and gray, respectively. E, shown are conformational changes of the oxidized and reduced Ahp1. The oxidized and reduced Ahp1 are colored in red and blue, respectively. The side chains of Cys-31, Cys-62, and Cys-120 are shown as sticks, and sulfur atoms are shown as yellow balls.

FIGURE 4.

Ahp1-Trx2 complex. A, shown is SDS-PAGE analysis of the complexes between Ahp1 mutants and Trx2C34S. The disulfide-linked complex could be reduced by DTT. Lanes 1–5, Ahp1C31S, Ahp1C62S, Trx2C34S, Ahp1C31S+Trx2C34S, and Ahp1C62S+Trx2C34S with 5 mm DTT; lane 6, protein marker; lanes 7–11, samples corresponding to lanes 1–5, respectively, without DTT. B, shown is the overall structure of disulfide-linked Ahp1C62S-Trx2C34S complex. The side chains of Cys-31 are labeled as sticks, and sulfur atoms are labeled as yellow balls. Ahp1 and Trx2 are colored cyan and pink, respectively. Residues of subunit B of Ahp1 are labeled with a prime, and residues of Trx2 are labeled with double prime. C, D, E are shown detailed representations of Ahp1-Trx2 complex interface. Hydrogen bonds (C), a disulfide bond (D), and hydrophobic interactions between subunit A of Ahp1 and subunit A of Trx2 (D). Hydrophobic interactions between subunit B of Ahp1 and subunit A of Trx2 (E). The involved residues are shown as sticks, and sulfur atoms of cysteines shown as yellow balls.

FIGURE 5.

A schematic diagram of Ahp1 catalytic cycle.