Metal distribution in Cu/Zn-superoxide dismutase revealed by native mass spectrometry

Michiko Tajiri, Hiroto Aoki, Atsuko Shintani, Kaori Sue, Satoko Akashi, Yoshiaki Furukawa

Research output: Contribution to journalArticlepeer-review

Abstract

Cu/Zn-superoxide dismutase (SOD1) is a homodimer with two identical subunits, each of which binds a copper and zinc ion in the native state. In contrast to such a text book case, SOD1 proteins purified in vitro or even in vivo have been often reported to bind a non-stoichiometric amount of the metal ions. Nonetheless, it is difficult to probe how those metal ions are distributed in the two identical subunits. By utilizing native mass spectrometry, we showed here that addition of a sub-stoichiometric copper/zinc ion to SOD1 led to the formation of a homodimer with a stochastic combination of the subunits binding 0, 1, and even 2 metal ions. We also found that the homodimer was able to bind four copper or four zinc ions, implying the binding of a copper and zinc ion at the canonical zinc and copper site, respectively. Such ambiguity in the metal quota and selectivity could be avoided when an intra-subunit disulfide bond in SOD1 was reduced before addition of the metal ions. Apo-SOD1 in the disulfide-reduced state was monomeric and was found to bind only one zinc ion per monomer. By binding a zinc ion, the disulfide-reduced SOD1 became conformationally compact and acquired the ability to dimerize. Based upon the results in vitro, we describe the pathway in vivo enabling SOD1 to bind copper and zinc ions with high accuracy in their quota and selectivity. A failure of correct metallation in SOD1 will also be discussed in relation to amyotrophic lateral sclerosis.

Original languageEnglish
Pages (from-to)60-68
Number of pages9
JournalFree Radical Biology and Medicine
Volume183
DOIs
Publication statusPublished - 2022 Apr

Keywords

  • Amyotrophic lateral sclerosis
  • Native mass spectrometry
  • SOD1
  • Thiol-disulfide

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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