Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant.
Mutations in copper-zinc superoxide dismutase (CuZnSOD) cause 25% of familial amyotrophic lateral sclerosis (FALS) cases. This paper examines one such mutant, H46R, which has no superoxide dismutase activity yet presumably retains the gain-of-function activity that leads to disease. We demonstrate that Cu(2+) does not bind to the copper-specific catalytic site of H46R CuZnSOD and that Cu(2+) competes with other metals for the zinc binding site. Most importantly, Cu(2+) was found to bind strongly to a surface residue near the dimer interface of H46R CuZnSOD. Cysteine was identified as the new binding site on the basis of multiple criteria including UV-vis spectroscopy, RR spectroscopy, and chemical derivatization. Cysteine 111 was pinpointed as the position of the reactive ligand by tryptic digestion of the modified protein and by mutational analysis. This solvent-exposed residue may play a role in the toxicity of this and other FALS CuZnSOD mutations. Furthermore, we propose that the two cysteine 111 residues, found on opposing subunits of the same dimeric enzyme, may provide a docking location for initial metal insertion during biosynthesis of wild-type CuZnSOD in vivo.
Liu
H
H
Department of Chemistry and Biochemistry, Pasarow Mass Spectrometry Laboratory, The Neuropsychiatric Institute, University of California, Los Angeles, CA, USA.
Zhu
H
H
Eggers
D K
DK
Nersissian
A M
AM
Faull
K F
KF
Goto
J J
JJ
Ai
J
J
Sanders-Loehr
J
J
Gralla
E B
EB
Valentine
J S
JS
D000690:Amyotrophic Lateral Sclerosis / Q000201:enzymology* / Q000235:genetics; D001120:Arginine / Q000378:metabolism; D001667:Binding, Competitive; D003035:Cobalt / Q000378:metabolism; D003300:Copper / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae; D012834:Silver / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D017186:Titrimetry
D003035:Cobalt; D012834:Silver; D006639:Histidine; D003300:Copper; D001120:Arginine; D013482:Superoxide Dismutase; D003545:Cysteine