The
human copper-binding protein metallothionein-3 (MT-3) can reduce
Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5–6 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.