The interaction between the Sco protein from Bacillus subtilis and copper
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Members of the Sco protein family have been proposed to function in the assembly of cytochrome c oxidase in the respiratory chain of all aerobic life forms. The Sco protein in Bacillus subtilis, BsSco, is characterized for its folding/unfolding behavior in the presence or absence of Cu(II) in this study. The folding/unfolding of apo-BsSco is investigated by CD and fluorescence spectroscopies. BsSco follows an apparent two-state mechanism in both folding and unfolding processes. The two apo forms of BsSco, reduced and oxidized, exhibit similar equilibrium stabilities suggesting that the formation of an intramolecular disulfide in oxidized apo-BsSco does not add to BsSco’s overall stability. In contrast, Cu(II) binding to reduced apo-BsSco results in extreme stabilization and resistance to unfolding in urea. However, when Cu(II) is present with unfolded, reduced apo-BsSco, the protein is rapidly oxidized. Another widely used denaturant, GdnHCl, is able to unfold Cu(II)-BsSco by allowing the loss of Cu(II) from the metal/protein complex. When the presence of Cu(II)-BsSco complex and the protein’s folded state are monitored simultaneously, the unfolding of Cu(II)-bound BsSco occurs coincidently with Cu(II) dissociation. We suggest that the loss of Cu(II) binding and the loss of BsSco’s native conformation are coincident, which leads to the conclusion that Cu(II)-BsSco does not unfold until it forfeits Cu(II). The kinetics of folding/unfolding of reduced, oxidized and Cu(II) bound BsSco are explored by stopped-flow fluorescence spectroscopy. The rate constants at which the two apo forms of BsSco fold and unfold are measured and plotted versus denaturant concentration. Reduced and oxidized forms of apo-BsSco are similar in folding and unfolding kinetics. Cu(II)-involved refolding kinetics of BsSco show that Cu(II) is able to accelerate the rate of refolding. However, the involvement of Cu(II) in the refolding process results in two competing processes: oxidation and Cu(II) binding. Which process predominates depends on the refolding rate which further depends on the denaturant concentration. This study has provided direct evidence for metal-involved stabilization of BsSco which is beneficial to efficiently fulfill its copper trafficking duty in a cellular environment.