Abstract
Immature copper-zinc superoxide dismutase (Sod1) is activated by its copper chaperone (Ccs1). Ccs1 delivers a single copper ion and catalyzes oxidation of an intra-subunit disulfide bond within each Sod1 monomer through a mechanistically ambiguous process. Here, we use residue specific fluorescent labeling of immature Sod1 to quantitate the thermodynamics of the Sod1•Ccs1 interaction while determining a more complete view of Ccs1 function. Ccs1 preferentially binds a completely immature form of Sod1 that is metal deficient and disulfide reduced (E, E-Sod1SH). However, binding induces structural changes that promote high-affinity zinc binding by the Ccs1-bound Sod1 molecule. This adds further support to the notion that Ccs1 likely plays dual chaperoning roles during the Sod1 maturation process. Further analysis reveals that in addition to the copper-dependent roles during Sod1 activation, the N- and C-terminal domains of Ccs1 also have synergistic roles in securing both Sod1 recognition and its own active conformation. These results provide new and measurable analyses of the molecular determinants guiding Ccs1-mediated Sod1 activation.
Highlights
Copper-zinc superoxide dismutase (Sod1) is an abundant antioxidant enzyme that readily converts superoxide anions into peroxide and molecular oxygen [2O2−+2H+ → H2O2+O2] [1]
An increasing amount of data points to metal deficient disulfide reduced (E, E-Sod1SH) forms of Sod1 mutants making up the majority of protein within the aggregates found in transgenic mice models and amyotrophic lateral sclerosis (ALS) patient autopsies [25,26,27,28,29]. These results strongly suggest that the Sod1/copper chaperone for Sod1 (Ccs1) transaction cannot be completed in these instances, though the point terminating the process may be different among the great variety of disease causing Sod1 mutants [7,9,30]
The labeled protein behaves essentially identical to the unlabeled disulfide-reduced Sod1 and shows no signs of aggregation or other non-native behavior in solution (Figure 2B)
Summary
Copper-zinc superoxide dismutase (Sod1) is an abundant antioxidant enzyme that readily converts superoxide anions into peroxide and molecular oxygen [2O2−+2H+ → H2O2+O2] [1]. Sod is subject to numerous post-translational modifications that convert the nascent polypeptide into its extremely stable homodimeric native conformation [2,3]. Immature Sod is targeted by the copper chaperone for Sod (Ccs1), which delivers the copper ion to the active site and catalyzes formation of an intra-subunit disulfide bond [4,5,6]. Zinc is assumed to be diffusively acquired by the environment and bound at the “zinc-site” adjacent to the active site [6,7,8]. Ccs has evolved an activity specific only for Sod and Sod cannot be activated by other copper chaperones [4,12]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
