Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster

Abstract

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)₄-Cys_5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O₂ and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn₇MT-3 and Cu(II) to form Cu(I)₄Zn(II)₄MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)₄-Cys₅ cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)₄-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O₂. We thus provide evidence that the MT-3 Cu(I)₄-Cys₅ cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Figure 1.

Reaction between Zn₇MT-3 and Cu(II) followed by stopped-flow electronic absorption spectroscopy. (a) Absorption spectra upon reaction of Zn₇MT-3 in 25 mM Tris-HCl/50 mM NaCl (10 μM) with Cu(II) (40 μM) at 37 °C, recorded every ms for the first 1 s, and 100 ms for the remaining 300 s. Inset: (top) Kinetic traces at 220 and 260 nm monitoring the fraction of bound Zn(II) or Cu(I), respectively, and corresponding fits with eq 1 (dotted line; for traces at 220 nm, t₁= 0.23 s, t₂ = 3.1 s, t₃= 75.6 s, R² = 0.9994; for traces at 260 nm, t₁= 0.31 s, t₂ = 2.8 s, t₃= 36.0 s, R² = 0.9996); (bottom) corresponding differential kinetics traces at 260 and 300 nm. The absolute differential absorption at time t is normalized over the total differential absorption at the completion of the reaction (ΔAbs_t/ΔAbs_max,, with t₀ = 0 s and t_f = 301 s). (b) Kinetic trace at 450 nm upon reaction of Zn₇MT-3 (100 μM) with Cu(II) (40 μM). Inset: Reconvoluted spectra kinetics between 320 and 600 nm in the initial part of the reaction (1 s; 10 ms steps up to 100 ms; 100 ms steps up to 1 s).

Figure 2.

Electronic absorption and luminescence spectroscopic characterization of the products of the reaction between Zn₇MT-3 and Cu(II) or Cu(I), in the presence and absence of O₂. (a) Electronic absorption spectra of Zn₇MT-3 (10 μM) and Cu₄Zn₄MT-3 (10 μM) generated by aerobic reaction of Zn₇MT-3 (10 μM) with Cu(II) (40 μM) incubated in air for 24 h. (b) 77 K luminescence emission spectrum of Cu₄Zn₄MT-3 (10 μM) generated by aerobic reaction of Zn₇MT-3 with Cu(II) and incubated in air for 24 h. (c) Electronic absorption spectra of Zn₇MT-3 (10 μM) and Cu₄Zn₄MT-3 (10 μM) generated by anaerobic reaction of Zn₇MT-3 with Cu(II) and incubated in N₂ atmosphere for 24 h. (d) 77 K luminescence emission spectrum of Cu₄Zn₄MT-3 (10 μM) generated by aerobic reaction of Zn₇MT-3 with Cu(II)incubated in air for 24 h. (e) Electronic absorption spectra of Zn₇MT-3 (10 μM) and Cu₄Zn₄MT-3 (10 μM) generated by anaerobic reaction of Zn₇MT-3 with Cu(I) and incubated in N₂ atmosphere for 24 h. (f) 77 K luminescence emission spectrum of Cu₄Zn₄MT-3 (10 μM) generated by anerobic reaction of Zn₇MT-3 with Cu(I) incubated in N₂ for 24 h.

Figure 3.

Time-dependent Cu(I)₄-thiolate cluster formation analyzed by low-temperature luminescence spectroscopy. (a) 77 K luminescence emission spectra of the product of the reaction between Zn₇MT-3 (72 μM) 40 μM Cu(II) upon freeze-quenching at short incubation times (1–250 s). (b) Determination of emission lifetimes at 425 and 575 nm after 1 s reaction, fitted using a single exponential decay function.

Figure 4.

DRA intermediates lifetimes and stability toward O₂ analyzed by stopped-flow electronic absorption spectroscopy. (a) Kinetic traces at 450 nm of the reaction of Zn₇MT-3 in 25 mM Tris-HCl/50 mM NaCl (10 μM) with Cu(II) (40 μM) at 37 °C, in the presence or absence of O₂. Inset: First 10 s of reaction. (b) Kinetic trace at 450 nm and corresponding fitting with a double exponential growth and decay function (eq 2 in Material and Methods) upon reaction between Zn₇MT-3 (72 μM) with Cu(II) (40 μM) at 25 °C. The observed decay constants (λ) and τ values are reported. (Inset) Experimental and kinetic trace at 450 nm in logarithmic base scale to highlight quality of the fit at ms time scales (R² = 0.9979; t_g1= 0.012 s; t_g2= 0.068 s; t_d1= 3 s; t_d2= 53 s).

Figure 5.

RFQ EPR characterization of the intermediates formed in the reaction between Zn₇MT-3 and Cu(II). X-band EPR spectra recorded at 15 K (a, c) and 30 K (b, d) upon rapid-mixing Zn₇MT-3 (225 μM) with Cu(II) (125 μM). Samples were quenched in liquid nitrogen at selected time points ranging from 1 to 1800 s. Quantitative simulations of 1 s RFQ sample at 15 K (c) and 30 K (d) were used to determine the contributions from Cu(II) and DRA species.

Figure 6.

Model for the reaction between Zn₇MT-3 and Cu(II) involving the formation of Cu-coupled disulfide radical anions. (a) Proposed Cu(II) reduction/Cu(I) binding to MT-3 via a long-lived disulfide radical anion-based mechanism. The scheme represents the redox reaction pathway for Cu(II)/Cu(I) and thiolate/disulfide couples and is adapted from Figure S1 (scheme 3) to highlight the existence of a novel Cu-coupled disulfide radical anion in the cluster assembly process revealed in this work. (b) Schematic representing the structure of the initial β-domain Zn(II)₃Cys₉ cluster and a model of the resulting Cu(I)₄Cys₅ cluster product obtained via metal exchange and Cu(II) reductions. The model of the Cu(I)₄-thiolate cluster is derived from spectroscopic data, as no atomic-resolution structural data are currently available.