Abstract
Rat heme-binding protein 23 (HBP23)/peroxiredoxin (Prx I) belongs to the 2-Cys peroxiredoxin type I family and exhibits peroxidase activity coupled with reduced thioredoxin (Trx) as an electron donor. We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. The C83S mutant was predominantly dimeric, in agreement with a previous crystallographic analysis (Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12333-12338). X-ray diffraction analysis of the decameric C52S mutant revealed a toroidal structure (diameter, approximately 130A; inside diameter, approximately 55A; thickness, approximately 45A). In contrast to human Prx I, which was recently reported to exist predominantly as the decamer with Cys(83)-Cys(83) disulfide bonds at all dimer-dimer interfaces, rat HBP23/Prx I has a Cys(83)-Cys(83) disulfide bond at only one dimer-dimer interface (S-S separation of approximately 2.1A), whereas the interactions at the other interfaces (mean S-S separation of 3.6A) appear to involve hydrophobic and van der Waals forces. This finding is consistent with gel filtration analyses showing that the protein readily interconverts between dimer and oligomeric forms. The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. At higher concentrations, where the protein was mostly decameric, less efficient attack of reduced Trx was observed in a [(14)C]iodoacetamide incorporation experiment. We suggest that the dimerdecamer interconversion may have a regulatory role.
Highlights
Peroxiredoxin (Prx)3 family proteins are found in all biological kingdoms from bacteria to animals and humans
Molecular Mass of heme-binding protein 23 (HBP23)/PrxI— we previously determined the crystal structure of the C83S mutant of rat liver HBP23/Prx I as a dimer (PDB entry 1QQ2), oligomerized forms other than the dimer are observed in solution for many 2-Cys Prxs from bacteria, plants, and humans [27, 28,35,36,37,38,39]
Comparison with molecular mass standards showed that HBP23/Prx I in the 20% homogenate was predominantly eluted at the position of molecular mass 44 kDa, but HBP23/Prx I in the 33% homogenate was eluted at the position of molecular mass 230 kDa, being present as both dimer and decamer
Summary
Peroxiredoxin; Trx, thioredoxin; HBP23, heme-binding protein 23; DTT, dithiothreitol; IAA, iodoacetamide; MIF, migration inhibitory factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PDB, Protein Data Bank. Human Prx I and Prx II have distinct quaternary structures; Prx I was recently reported to exist predominantly as the decamer with a Cys83-Cys disulfide bridge at the dimer-dimer interface, whereas Prx II exists as a dimer, because the corresponding Cys residue is replaced with Thr in Prx II [40]. Based on this feature, it was proposed that the function of Prx I is distinct from that of Prx II. In contrast to the recently reported result for human Prx I [40], our crystal structure of the decameric rat C52S mutant (Protein Data Bank (PDB) entry 2Z9S) showed that only one out of the five dimer-dimer interfaces involved a disulfide bridge from Cys to Cys of the adjacent dimer, indicating that stabilization of the decamer structure is not due to Cys83-Cys disulfide bridges at the dimer-dimer interfaces, but rather, is predominantly due to hydrophobic interaction and structural fitting, as is seen in the decameric structures of bacterial (26 –28) and human [25] Prx II
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