Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site

Abstract

Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed "entry site" for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.

The yeast copper chaperone for copper-zinc superoxide dismutase (CCS1) is a multifunctional chaperone promoting all levels of SOD1 maturation

“…The second domain (D2, residues 78 -216) possesses sequence and structural homology with SOD1 (21), suggesting that it recognizes SOD1 by mimicking SOD1⅐SOD1 homodimeric interactions. Supporting this notion, structures of yeast CCS1 alone (21) and in complex with yeast SOD1 (24,25) reveal D2-mediated interactions with SOD1. Mutations compromising the CCS1 D2 interface abrogate CCS1-mediated SOD1 activation (26).…”

“…Our recently published SOD1⅐CCS1 heterocomplex structure reveals a previously unobserved ␤-hairpin conformation of D3 that is stabilized by interactions with residues from SOD1, CCS1 D2, and the linker region between D1 and D2 (25). This novel conformer places the conserved CXC motif near the heterodimeric interface and creates an entry site for Cu delivery during SOD1 activation (25).…”

“…Our recently published SOD1⅐CCS1 heterocomplex structure reveals a previously unobserved ␤-hairpin conformation of D3 that is stabilized by interactions with residues from SOD1, CCS1 D2, and the linker region between D1 and D2 (25). This novel conformer places the conserved CXC motif near the heterodimeric interface and creates an entry site for Cu delivery during SOD1 activation (25). Further analysis of the SOD1⅐ CCS1 heterodimeric structure is suggestive of a possible 180°r otation of the D3 ␤-hairpin to transfer Cu(I) from the MXCXXC site in D1 to the SOD1⅐CCS1 "entry site," as suggested previously (28).…”

The molecular pathogenesis of superoxide dismutase 1-linked ALS is promoted by low oxygen tension

“…Oxidation of the C57-C146 disulfide bond is catalyzed by CCS (Culotta et al, 1997, Fetherolf et al, 2017, Furukawa et al, 2004), and it can be reduced by a mechanism involving reducing equivalents from GSH via glutaredoxin-1 (Carroll et al, 2006). To determine whether these factors were affected by low O 2 tension and responsible for increased misfolding, we analyzed their levels in MNACs grown at 19% vs 2% O 2 .…”

“…If the C57-C146 disulfide bond in SOD1 were in equilibrium with reduced and oxidized glutathione (GSH/GSSG) -the principal redox couple in the cytosol - it would be almost completely reduced (Mercatelli et al, 2016, Schwarzlander et al, 2016). Instead, the status of C57-C146 seems to be determined kinetically with oxidation provided by O 2 and catalyzed by copper chaperone for SOD (CCS) (Culotta et al, 1997, Fetherolf et al, 2017, Furukawa et al, 2004). Reduction is provided by glutaredoxin-1 using reducing equivalents from GSH (Carroll et al, 2006).…”