Research ArticleSTRUCTURAL BIOLOGY

Structural basis and mechanism for metallochaperone-assisted assembly of the CuA center in cytochrome oxidase

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Science Advances  31 Jul 2019:
Vol. 5, no. 7, eaaw8478
DOI: 10.1126/sciadv.aaw8478
  • Fig. 1 X-ray structures of the ScoI·Cu2+ and CoxB·CuA holo-proteins, and formation of the ScoI·Cu2+·CoxB complex.

    (A and B) Cartoon representations of the 1.4-Å structure of the periplasmic ScoI domain (18.1 kDa) with bound Cu2+ (A) and the 1.3-Å structure of the periplasmic CoxB domain (15.6 kDa) harboring the CuA center (B). Top, overall folds; bottom, coordination spheres around the bound copper ions (orange). Residues coordinating Cu are shown as stick models, and distances between the liganding atoms and the Cu ions are given. (C) Cw EPR spectra of ScoI·Cu2+ (red) and the ScoI·Cu2+·CoxB complex (dark gray), normalized to identical concentration. (D) Cw EPR spectrum of CoxB·CuA. (E) Analysis of ScoI·Cu2+·CoxB complex (CP) formation at pH 7.0 and 25°C by gel filtration. CoxB (40 μM) was titrated with the indicated amounts of ScoI·Cu2+. (F) Titration of 15 μM CoxB with ScoI·Cu2+ at pH 7.0 and 25°C, recorded via the ScoI·Cu2+·CoxB–specific absorption at 520 nm. The sharp kink at the 1:1 ratio indicates that ScoI·Cu2+·CoxB is a high-affinity complex with a dissociation constant below 10−7 M. (G) Stopped-flow absorbance kinetics of ScoI·Cu2+·CoxB complex formation at 25°C and pH 7.0, recorded at 520 nm. CoxB (25 μM) was mixed with a 1.5-, 3-, or 5-fold excess of ScoI·Cu2+. Absorbance traces (dotted lines) were fitted globally (solid lines) to a mechanism assuming formation of an encounter complex (association: k1 = 1.5 ± 0.014 × 105 M−1 s−1; dissociation: k2 = 1.68 ± 0.04 s−1) followed by intramolecular rearrangement to the final complex (k3 = 1.20 ± 0.01 s−1).

  • Fig. 2 Copper-binding properties of PcuC.

    (A) ESI mass spectra of apo-PcuC (blue), PcuC·Cu1+·Cu1+ (pink), and PcuC·Cu2+·Cu2+ (violet). (B) EPR spectra (continuous wave, 9.5 GHz, 40 K), normalized to identical protein concentration, of PcuC·Cu2+·Cu2+ (violet), PcuC·Cu1+·Cu1+ in the presence of 50 mM Na-dithionite (gray), PcuC·Cu1+·Cu1+ in the absence of Na-dithionite (pink), and PcuC after incubation with 1.5 (green) or 3 meq (blue) of a Cu1+/Cu2+ mixture (1:1). Copper complexes were prepared under anaerobic conditions, and excess Cu ions were removed by buffer exchange with 20% glycerol, 20 mM Mops-NaOH (pH 7.0), and 50 mM NaCl before EPR measurements. (C) EPR determination of distances between the Cu2+ ions in PcuC·Cu2+·Cu2+ by four-pulse ultra-wideband DEER, with uncertainties within 15% root mean square deviation. The DEER distance distribution shows Cu(II)-Cu(II) interspin distances widely distributed with a full width at half maximum (FWHM) ranging from 1.6 to 2.6 nm. The small peak observed in the 3- to 4-nm distance region is at the edge of resolution for the achievable dipolar evolution time of 1.4 μs (fig. S3), is therefore affected by trace length and noise, and was not interpreted.

  • Fig. 3 Differential binding of Cu1+ and Cu2+to PcuC studied by NMR.

    (A to F) Overlays of 2D-[13C,1H]-HMQC NMR spectra of Met-(methyl-13C)–labeled apo-PcuC and PcuC loaded with Cu1+ and/or Cu2+. Cartoons of PcuC are shown at the bottom of each panel, depicting the core domain (filled oval) and the flexible C-terminal extension (curved line). Cu1+ and Cu2+ are represented by yellow and orange balls, respectively. The colored circles around the cartoons indicate the color of the spectrum of each species. (A) C-terminally truncated PcuC (apo-PcuC ΔC) was used for identifying methionine signals from the flexibly disordered C-terminal segment (“C-term”) and the structured core domain. (B) Upon addition of 1 meq of Cu1+ to PcuC, only signals from the core domain shift, indicating preferential Cu1+ binding to the core domain. (C) Cu2+ binds to the C-terminal extension of PcuC, evidenced by the quenching of the signals from this region due to the paramagnetic properties of Cu2+. (D) In the presence of excess Cu1+, shifts of the methionine peaks in both the core and C-terminal extension indicate binding of Cu1+ ions to both sites. The sample contained 50 mM sodium dithionite to suppress Cu1+ oxidation. (E) In presence of 1.5 meq of both Cu1+ and Cu2+, Cu1+ preferentially binds to the conserved, primary Cu1+-binding site in the PcuC core. (F) Same sample of PcuC·Cu1+·Cu1+ as in (D) but in the absence of sodium dithionite. The Cu1+ bound at the C terminus became oxidized to Cu2+, leading to quenching of the C-terminal methionine peaks. In contrast, the Cu1+ bound to the PcuC core stayed resistant against oxidation.

  • Fig. 4 Neither apo-PcuC nor PcuC·Cu1+ can release CoxB from the ScoI·Cu2+·CoxB complex.

    (A) SDS-polyacrylamide gel (20%) of the purified protein components used in the titration experiments shown here and in Fig. 5. (C to E) Titration of ScoI·Cu2+·CoxB with apo-PcuC or PcuC·Cu1+ at pH 7.0 and 25°C, analyzed by analytical gel filtration. Eluted proteins were detected via their absorbance at 280 nm. The ScoI·Cu2+·CoxB complex (20 μM) was kept constant in all titration experiments and mixed with 0.25 to 3 meq of apo-PcuC or PcuC·Cu1+, and the reaction products were separated by gel filtration. (B) Gel filtration profiles of the individual components and controls used in the titration experiments shown here and in Figs. 1E and 5, demonstrating that (i) PcuC and ScoI exhibit the same retention times but can be well separated from ScoI·Cu2+·CoxB and CoxB and (ii) no complex is formed between apo-ScoI and CoxB and between ScoI·Cu2+ and oxidized CoxB (CoxBox). (C) Titration of ScoI·Cu2+·CoxB with apo-PcuC. Neither dissociation of ScoI·Cu2+·CoxB nor CoxB·CuA formation could be observed. (D) Titration of ScoI·Cu2+·CoxB with PcuC·Cu1+, showing that only a tiny fraction of ScoI·Cu2+·CoxB dissociated at high excess of PcuC·Cu1+. (E) Titration experiment from (D) but with protein detection at the CoxB·CuA–specific absorbance maximum of 813 nm instead of 280 nm, showing that no CoxB·CuA was formed. Purified CoxB·CuA (20 μM) was used as reference for 100% CoxB·CuA formation (magenta peak).

  • Fig. 5 Two equivalents of PcuC·Cu1+·Cu2+ are required for conversion of ScoI·Cu2+·CoxB to CoxB·CuA.

    (A) Mechanism of in vitro formation of CoxB·CuA (97% yield), deduced from the experiments shown in (B to H). (B and F) Titration of ScoI·Cu2+·CoxB (20 μM) with PcuC·Cu1+·Cu2+ at pH 7.0 and 25°C, analyzed by gel filtration (protein detection at 280 nm). ScoI·Cu2+·CoxB dissociation was completed after addition of one PcuC·Cu1+·Cu2+ equivalent. (C) Gel filtration runs from (B) but with protein detection at the CoxB·CuA–specific absorbance maximum at 813 nm. CoxB·CuA formation only reached its maximum after addition of two PcuC·Cu1+·Cu2+ equivalents to ScoI·Cu2+·CoxB. (D) Absorption spectra of the isolated CoxB·CuA peaks from (B) and (C), showing that two equivalents of PcuC·Cu1+·Cu2+ added to ScoI·Cu2+·CoxB quantitatively reconstituted the CoxB·CuA center (absorbance maxima at 367, 479, and 813 nm). (E) CoxB·CuA peak areas from (C) plotted against the added equivalents of PcuC·Cu1+·Cu2+. (G) Stopped-flow absorbance kinetics of the dissociation of the ScoI·Cu2+·CoxB complex (25 μM) by 1 equivalent (25 μM) of PcuC·Cu1+·Cu2+, yielding a second-order rate constant of 6.83 ± 0.02 × 102 M−1 s−1 (see Materials and Methods for the details). (H) Detection of cytochrome oxidase activity in B. diazoefficiens wild type (WT; positive control) and deletion mutants deficient in ScoI or PcuC. A coxB deletion mutant served as a negative control. Bacteria were grown aerobically in peptone–salt–yeast extract medium with or without 50 μM CuCl2. Identical amounts of cells were spotted onto a filter paper soaked with the indicator dye TMPD that reacts to indophenol blue when CoxB is active.

  • Fig. 6 Model for copper trafficking to the CoxB subunit of aa3-type cytochrome c oxidase in the periplasm of B. diazoefficiens.

    CuA center formation in subunit II (CoxB) of aa3 oxidase of B. diazoefficiens requires soluble periplasmic PcuC (violet) and membrane-anchored ScoI (red). The periplasmic thioredoxin-like reductase TlpA maintains the active-site cysteine pairs of ScoI and CoxB in the reduced (dithiol) state that is required for Cu2+ binding. The Cu2+-specific chaperone ScoI readily reacts with apo-CoxB to a stable ScoI·Cu2+·CoxB complex (reaction I). The preferred metalation state of PcuC has a Cu1+ ion bound to the folded PcuC core domain and a Cu2+ ion bound to its flexible, C-terminal extension. PcuC·Cu1+·Cu2+ specifically reacts with the ScoI·Cu2+·CoxB complex and releases CoxB·Cu2+ from the complex (reaction II). A second equivalent of PcuC·Cu1+·Cu2+ then transfers Cu1+ to CoxB·Cu2+, and the CoxB·CuA center is formed (reaction III). As only Cu ions bound to the C-terminal PcuC extension can be transferred to CoxB, donation of Cu1+ to CoxB·Cu2+ requires intramolecular transfer of an electron from Cu1+ in the core domain to Cu2+ at the C-terminal PcuC extension. The overall reaction scheme at the bottom shows that, formally, ScoI·Cu2+ is not consumed in ScoI/PcuC-mediated CoxB·CuA biogenesis, indicating that catalytic amounts of ScoI relative to CoxB might be sufficient for efficient CoxB·CuA center formation in vivo.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw8478/DC1

    Fig. S1. Structural characterization of ScoIox, ScoI·Cu2+, CoxB·CuA, and PcuC.

    Fig. S2. Amino acid sequence alignment of PcuC homologs.

    Fig. S3. EPR determination of distances between two Cu2+ ions in PcuC·Cu2+·Cu2+.

    Fig. S4. NMR experiments showing that the C-terminally truncated PcuC variant PcuC ΔC can also bind Cu2+.

    Fig. S5. Investigation of the Cu2+-binding properties of the C-terminal PcuC peptide by NMR.

    Fig. S6. Absorbance spectra of ScoI·Cu2+·CoxB, CoxB·CuA, ScoI·Cu2+, and PcuC·Cu1+·Cu2+.

    Fig. S7. Equilibrium competition between PcuC or ScoI and BCS for binding of Cu1+ reveals a specific high-affinity Cu1+-binding site in the PcuC core domain and a low-affinity Cu1+-binding site in the C-terminal PcuC extension and demonstrates that ScoI does not bind Cu1+.

    Fig. S8. Detection of cytochrome oxidase activity in B. diazoefficiens mutants under copper starvation.

    Fig. S9. NMR analysis of Cu transfer from PcuC·Cu1+·Cu2+ to the ScoI·Cu2+·CoxB complex.

    Fig. S10. Alternative, but disfavored, mechanism of CoxB·CuA formation from CoxB·Cu2+ and only one equivalent of PcuC·Cu1+·Cu2+.

    Fig. S11. Cw EPR spectra of the PcuC·Cu1+·Cu2+ + ScoI·Cu2+·CoxB reaction mixture.

    Fig. S12. The ScoI·Cu2+ complex shows limited long-term stability due to Cu2+-induced formation of a disulfide bond between the active-site cysteines of ScoI that causes release of Cu2+.

    Fig. S13. CoxB·CuA formation from apo-CoxB and PcuC·Cu1+·Cu2+ in the absence of ScoI.

    Table S1. X-ray data collection and refinement statistics (molecular replacement).

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Structural characterization of ScoIox, ScoI·Cu2+, CoxB·CuA, and PcuC.
    • Fig. S2. Amino acid sequence alignment of PcuC homologs.
    • Fig. S3. EPR determination of distances between two Cu2+ ions in PcuC·Cu2+·Cu2+.
    • Fig. S4. NMR experiments showing that the C-terminally truncated PcuC variant PcuC ΔC can also bind Cu2+.
    • Fig. S5. Investigation of the Cu2+-binding properties of the C-terminal PcuC peptide by NMR.
    • Fig. S6. Absorbance spectra of ScoI·Cu2+·CoxB, CoxB·CuA, ScoI·Cu2+, and PcuC·Cu1+·Cu2+.
    • Fig. S7. Equilibrium competition between PcuC or ScoI and BCS for binding of Cu1+ reveals a specific high-affinity Cu1+-binding site in the PcuC core domain and a low-affinity Cu1+-binding site in the C-terminal PcuC extension and demonstrates that ScoI does not bind Cu1+.
    • Fig. S8. Detection of cytochrome oxidase activity in B. diazoefficiens mutants under copper starvation.
    • Fig. S9. NMR analysis of Cu transfer from PcuC·Cu1+·Cu2+ to the ScoI·Cu2+·CoxB complex.
    • Fig. S10. Alternative, but disfavored, mechanism of CoxB·CuA formation from CoxB·Cu2+ and only one equivalent of PcuC·Cu1+·Cu2+.
    • Fig. S11. Cw EPR spectra of the PcuC·Cu1+·Cu2+ + ScoI·Cu2+·CoxB reaction mixture.
    • Fig. S12. The ScoI·Cu2+ complex shows limited long-term stability due to Cu2+-induced formation of a disulfide bond between the active-site cysteines of ScoI that causes release of Cu2+.
    • Fig. S13. CoxB·CuA formation from apo-CoxB and PcuC·Cu1+·Cu2+ in the absence of ScoI.
    • Table S1. X-ray data collection and refinement statistics (molecular replacement).

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