Copper Stabilizes a Heterodimer of the yCCS Metallochaperone and Its Target Superoxide Dismutase

The copper chaperone for superoxide dismutase (CCS) activates the antioxidant enzyme Cu,Zn-SOD (SOD1) by directly inserting the copper cofactor into the apo form of SOD1. Neither the mechanism of protein-protein recognition nor of metal transfer is clear. The metal transfer step has been proposed to...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2001-10, Vol.276 (42), p.38410-38416
Main Authors: Torres, Andrew S., Petri, Victoria, Rae, Tracey D., O'Halloran, Thomas V.
Format: Article
Language:English
Subjects:
Anisotropy
Binding Sites
Blotting, Western
Chromatography, Gel
Copper - chemistry
Copper - metabolism
Dimerization
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Histidine - chemistry
Molecular Chaperones - chemistry
Molecular Chaperones - metabolism
Mutation
Phenylalanine - metabolism
Precipitin Tests
Protein Binding
Saccharomyces cerevisiae Proteins
Superoxide Dismutase - genetics
Superoxide Dismutase - metabolism
Time Factors
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The copper chaperone for superoxide dismutase (CCS) activates the antioxidant enzyme Cu,Zn-SOD (SOD1) by directly inserting the copper cofactor into the apo form of SOD1. Neither the mechanism of protein-protein recognition nor of metal transfer is clear. The metal transfer step has been proposed to occur within a transient copper donor/acceptor complex that is either a heterodimer or heterotetramer (i.e. a dimer of dimers). To determine the nature of this intermediate, we generated a mutant form of SOD1 by replacing a copper binding residue His-48 with phenylalanine. This protein cannot accept copper from CCS but does form a stable complex with apo- and Cu-CCS, as observed by immunoprecipitation and native gel electrophoresis. Fluorescence anisotropy measurements corroborate the formation of this species and further indicate that copper enhances the stability of the dimer by an order of magnitude. The copper form of the heterodimer was isolated by gel filtration chromatography and contains one copper and one zinc atom per heterodimer. These results support a mechanism for copper transfer in which CCS and SOD1 dock via their highly conserved dimer interfaces in a manner that precisely orients the Cys-rich copper donor sites of CCS and the His-rich acceptor sites of SOD1 to form a copper-bridged intermediate.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M104790200