期刊
MOLECULAR MICROBIOLOGY
卷 119, 期 4, 页码 423-438出版社
WILEY
DOI: 10.1111/mmi.15032
关键词
copper toxicity; metal efflux pumps; thiol oxidation
Copper can activate disulfide bonds in proteins, but its efficiency as a catalyst is limited. This study found that copper oxidizes thiol N-acetylcysteine, but the slow regeneration of copper(II) limits the efficiency of thiol oxidation. Copper overload in cells impairs growth and inactivates enzymes, but it does not activate certain transcription factors. Overall, copper accumulation is mostly found in the Cu(I) valence, impacting few thiol-containing proteins in the periplasm.
Copper avidly binds thiols and is redox active, and it follows that one element of copper toxicity may be the generation of undesirable disulfide bonds in proteins. In the present study, copper oxidized the model thiol N-acetylcysteine in vitro. Alkaline phosphatase (AP) requires disulfide bonds for activity, and copper activated reduced AP both in vitro and when it was expressed in the periplasm of mutants lacking their native disulfide-generating system. However, AP was not activated when it was expressed in the cytoplasm of copper-overloaded cells. Similarly, this copper stress failed to activate OxyR, a transcription factor that responds to the creation of a disulfide bond. The elimination of cellular disulfide-reducing systems did not change these results. Nevertheless, in these cells, the cytoplasmic copper concentration was high enough to impair growth and completely inactivate enzymes with solvent-exposed [4Fe-4S] clusters. Experiments with N-acetylcysteine determined that the efficiency of thiol oxidation is limited by the sluggish pace at which oxygen regenerates copper(II) through oxidation of the thiyl radical-Cu(I) complex. We conclude that this slow step makes copper too inefficient a catalyst to create disulfide stress in the thiol-rich cytoplasm, but it can still impact the few thiol-containing proteins in the periplasm. It also ensures that copper accumulates intracellularly in the Cu(I) valence.
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