Probing the Kinetics and Thermodynamics of Copper(II) Binding toBacillus subtilisSco, a Protein Involved in the Assembly of the Cu_ACenter of CytochromecOxidase

Abstract

BsSco is a member of the Sco protein family involved in the assembly of the Cu(A) center within cytochrome c oxidase. BsSco forms a complex with Cu(II) that has properties consistent with dithiolate ligation. Stopped-flow UV-visible absorbance and fluorescence coupled with multiwavelength analysis reveal biphasic binding kinetics between BsSco and Cu(II). An initial species appears with absorbance centered at 382 nm at a copper concentration-dependent rate (2.9 x 10(4) M(-1) s(-1)). The initial species decays at a first-order rate (1.5 s(-1)) to the equilibrium form with a maximum at 352 nm. Formation of the BsSco-Cu(II) complex is accompanied by quenching of protein fluorescence. The copper concentration-dependent phase gives 70% of the total quenching, while the final 30% develops during the second phase of the absorbance change. The pH dependence of copper binding shows that the copper-dependent rate increases by 50-fold as the pH decreases from 8.5 to 5.5 with an apparent pK(a) of 6.7. The slower phase rate is independent of pH. Comparison of circular dichroism spectra between apo-BsSco and the BsSco-Cu(II) complex reveals a small change in the UV region consistent with a subtle conformational change upon copper binding. There is formation of a distinctive visible CD spectrum in the BsSco-Cu(II) complex. A model is presented in which the kinetic and thermodynamic stability of the BsSco-Cu(II) complex results from a two-step mechanism. Release of copper would be facilitated in the intermediate form of BsSco, and attaining such a low-Cu(II) affinity state may be important for BsSco's function in Cu(A) assembly.

Disparate Pathways for the Biogenesis of Cytochrome Oxidases in Bradyrhizobium japonicum

“…All of these results are consistent with the purported role of the eukaryotic (i.e. mitochondrial) ScoI-like proteins (19,22,71,72) and some prokaryotic ScoI homologs (20,23,73) in the biogenesis of the membrane-peripheral, Cu A -containing subunit II domain. Akin to Cox11/CtaG/CoxG, however, the direct transfer of copper from the ScoI chaperone to the target subunit remains to be demonstrated experimentally.…”

A Copper Relay System Involving Two Periplasmic Chaperones Drives cbb3-Type Cytochrome c Oxidase Biogenesis in Rhodobacter capsulatus

“…10,30,31 The affinities of R. capsulatus SenC and PccA for Cu(I) were determined using a competition assay with bathocuproine disulfonate (BCS) as described previously 29,32 (Figure 4b). Briefly, 10 μ M Cu(II) was reduced with 10 μ M ascorbic acid and incubated with 25 μ M BCS, and the [Cu I (BCS) 2 ] 3− complex (Cu-BCS) thus formed was titrated with different SenC or PccA concentrations varying from 0 to 20 μ M. The decrease in Cu-BCS with increasing total protein concentration was monitored at 483 nm, and the competition $K_{\mathrm{normalD}}^{\text{Cu}(\mathrm{I})}$ values were determined as described in the Supporting Information.…”

“…29,33 An exception is the Bacillus subtilis ScoI homologue, which seems to have approximately picomolar affinity for Cu(II), but only approximately micromolar affinity for Cu(I). 31,34 The basis of this difference is unclear, although the Gram-positive bacterium B. subtilis lacks a PccA homologue that could transfer Cu(I) to ScoI. Perhaps preferential binding of Cu(II) by B. subtilis ScoI in an oxidizing environment, such as the extracellular face of the membrane, might be beneficial in this case.…”

Copper Metallochaperones

“…The intriguing aspect of the Bacillus Sco1 is its dramatic preference for Cu(II) over Cu(I) (98). The marked stability of the Cu(II)-Sco1 complex may necessitate a conversion step to an intermediate state of Cu(I)-Sco1 compatible with Cu transfer (99). …”