There is increasing evidence that H2O2 serves as an intracellular messenger mediating various cell functions including proliferation, differentiation, apoptosis, and senescence, when produced in low amount and in a controlled fashion. Understanding the intracellular messenger function of H2O2 calls for studies of how receptor occupation elicits the production of H2O2 and how H2O2 is eliminated after the completion of its mission. The generation of H2O2 in cells stimulated with growth factors requires the activation of phosphatidylinositol 3-kinase (PI3K) and the small GTPase Rac. We demonstrated that both betaPix, a guanine nucleotide exchange factor for Rac (Rac-GEF), and Nox1, a protein related to gp91phox (Nox2) of phagocytic cells, contribute to the growth factor-induced production of H2O2 in nonphagocytic cell lines. BetaPix was shown to be constitutively associated with the COOH-terminal region of Nox1, whereas this region of Nox1 did not bind Vav1, another Rac-GEF. Rac1 was also shown to bind to the COOH-terminal region of Nox1 in a growth factor-dependent manner. Both growth factor-induced Rac1 activation and H2O2 production were completely blocked in cells depleted of betaPix by RNA interference. The pleckstrin homology and leucine zipper domains of betaPix, which mediate bPix activation by products of PI3K action and betaPix homodimerization, respectively, were essential for growth factor-induced H2O2 production. Moreover, expression of a COOH-terminal fragment of Nox1 completely inhibited growth factor-induced H2O2 generation. These results suggest that H2O2 production in growth factor-stimulated cells is mediated by the sequential activation of PI3K, betaPix, and Rac1, the latter of which then binds to Nox1 to stimulate electron flow from NADPH to oxygen molecules. Peroxiredoxin I (Prx I) catalyzes the reduction of H2O2 with the use of reducing equivalents derived from NADPH through thioredoxin (Trx) and Trx reductase. The catalytic cycle of this dimeric enzyme includes the specific oxidation of Cys51-SH by H2O2 to Cys51-SOH, the formation of a disulfide between the Cys51-sulfenic acid and Cys172-SH of the paired subunit, and the reduction of the intermolecular disulfide by Trx. By following Prx-dependent NADPH oxidation spectrophotometrically, we observed that Prx activity decreases gradually with time. The decay in activity was coincident with the conversion of Prx I to a more acidic species as assessed by 2-dimensional gel electrophoresis. Mass spectral analysis and studies with Cys mutants determined that this shift in pI was due to selective oxidation of the catalytic site Cys51-SH to Cys-SOOH. Thus, the sulfenic Cys51 generated as an intermediate during catalysis appeared to undergo occasional further oxidation to the sulfinic state, which cannot be reversed by Trx. The presence of H2O2 alone was not sufficient to cause oxidation of the active site Cys to sulfnic acid. Rather, the presence of complete catalytic components (H2O2, Trx, Trx reductase, and NADPH) was necessary, indicating that such hyper-oxidation occurred only when Prx I was engaged in the catalytic cycle. Likewise, a mutant Prx I, in which Cys172 was replaced by Ser, did not undergo hyper-oxidation in the presence of a complete reaction mixture because the mutant was not catalytically active. Hyper-oxidation of the mutant enzyme required not only H2O2 but also a catalysis-supporting thiol, dithiothreitol. Kinetic analysis of Prx I inactivation in the presence of a steady-state, low level (<1 microM) of H2O2 indicated that Prx I was hyper-oxidized at a rate of 0.072% per turnover at 30?C. Hyper-oxidized Prx I was detected in HeLa cells cultured under normal conditions and increased in a concentration dependent-manner when H2O2 was applied to the culture media. Such hyper-oxidation is likely to result in H2O2 accumulation and thereby contribute to cell death. Previously, we reported that Prx purified from yeast is readily inactivated during catalysis (1). We speculated that such inactivation occurred if the sulfenic acid moiety of the reaction intermediate was further oxidized by H2O2 to sulfinic acid (Cys- SO3H) before disulfide formation with Cys172 could occur (1). Sulfinic acid cannot be reduced by the Trx or DTT included in the assay mixture. Recently Mitsumoto et al. (29) used two-dimensional polyacrylamide gel electrophoresis (2-D gel) to compare proteins in human umbilical vein endothelial cells before and after exposure of cells to H2O2. In H2O2-treated cells, a number of proteins including Prx I and Prx II demonstrated altered migration consistent with decreased isoelectric pH (pI), suggesting that such oxidative inactivation might also occur in cells. However, these acidic Prx enzymes were not characterized in detail. We have now investigated the mechanism of human Prx I inactivation by H2O2. Here, we demonstrated that the enzymatic inactivation and concomitant acidic shift of Prx on 2-D gels are due in fact to the conversion of the active site cysteine to sulfinic acid (Cys- SO2H). Furthermore, we observed that only those Prx molecules actively engaged in the catalytic cycle are vulnerable to oxidative inactivation.
