PMC

Proposed mechanism of the O₂-dependent post-translational modification of ySOD1 by Cu-yCCS (see text). The inset shows the heterodimeric structure between H48F ySOD1 and yCCS (1JK9) (). The green residues indicate the copper-binding site of ySOD1, and the intermolecular disulfide bond is represented as a yellow stick.

(A) Western blotting of in situ modification of ySOD1 with AMS using the cell lysate. Lane 1: E,Zn-ySOD1^S–S; lane 2: E,Zn-ySOD1^SH modified with AMS. Lanes 3 and 5 are the cell lysate of SY1699 and ccs1Δ, respectively, while the lysate after modification with AMS is shown in lanes 4 (SY1699) and 6 (ccs1Δ). The samples were loaded on 12.5% SDS–PAGE under nonreducing conditions. (B) Comparison of the ySOD1 band mobility on SDS–PAGE. The samples are as follows: lane 1, E,Zn-ySOD1^SH; lane 2, E,Zn-ySOD1^S–S; lane 3, C146S-ySOD1; lane 4, E,Zn-ySOD1^SH loaded with β-ME.

Western blotting of the ySOD1 multimer formed by addition of H₂O₂. E,E-ySOD1^SH (10 μM) was aerobically incubated with or without 100 μM H₂O₂ for an hour, as indicated. After modification with AMS, the protein solutions were mixed with the sample buffer (A) without or (B) with β-ME and loaded on 12.5% SDS–PAGE. Each band position of monomeric species is tentatively assigned on the basis of the apparent electrophoretic mobility.

(A) Effects of the dissolved oxygen on the disulfide formation in E,Zn-ySOD1^SH. Protein was incubated for the given time at 37°C, and following modification with AMS, 2 μg of ySOD1 was loaded on the 12.5% SDS–PAGE under nonreducing conditions. (B) The fraction of the reduced form plotted against the incubation time: the time course of the disulfide formation in 13 μM E,Zn-ySOD1^SH with equimolar (□) CuSO₄, (▪) CuSO₄ in 100 μM EDTA, (○) Cu(CH₃CN)₄(PF₆), and (•) Cu(CH₃CN)₄(PF₆) in 100 μM BCS. The results of aerobic incubation of 13 μM E,Zn-ySOD1^SH (▴) are also shown. (Inset) The time course of the disulfide formation in 13 μM E,Zn-ySOD1^SH with 5 μM Cu,Zn-ySOD1^S–S.

After 13 μM E,Zn-ySOD1^SH or E,Zn-ySOD1^S–S was incubated with equimolar Cu-yCCS or CuSO₄, about 200 ng of the samples was loaded on (A) 10% NATIVE-PAGE gel for in-gel assay and (B) 12.5% SDS–PAGE gel under reducing conditions for Western blotting analysis. (C) The gel filtration chromatograms of E,Zn-ySOD1^S–S and E,Zn-ySOD1^SH. (D) MALDI-TOF mass spectra of the heterodimer: C146S ySOD1 (m/z^calc=15 707, m/z^obs=15 708), Cu-yCCS (m/z^calc=27 198, m/z^obs=27 192), and C146S/yCCS heterodimer (m/z^calc=42 905, m/z^obs=42 921). Peaks denoted by ^* are from +2-charged states of the proteins. (Inset) The formation of the disulfide-linked heterodimer was further examined using 12.5% SDS–PAGE. Molecular marker (lane 1) and E,Zn-C146S ySOD1 aerobically incubated for an hour with Cu-yCCS at 37°C, loaded (lane 2) with or (lane 3) without β-ME, are shown.

The in-gel assay of the SOD1 activity: E,Zn-ySOD1^SH WT (lanes 1–3) and C146S (lanes 4–6). Reaction mixture contained 13 μM ySOD1, 100 μM EDTA, and 100 μM BCS in 50 mM HEPES, pH 7.2, with or without 13 μM Cu-yCCS and was prepared in a glove box (lanes 1, 2, 4, and 5). In lanes 3 and 6, the solution contains 13 μM CuSO₄ without EDTA and BCS. After aerobic incubation at 37°C for an hour, an aliquot containing 200 ng of ySOD1 was applied to (A) 10% NATIVE-PAGE for the activity assay or (B) 12.5% SDS–PAGE under reducing conditions for Western blotting. (C) The thiol-disulfide status was also checked with 12.5% SDS–PAGE after modification with AMS.

(A) Disulfide formation in E,Zn-ySOD1^SH with Cu-yCCS. The reaction mixture contains 13 μM E,Zn-ySOD1^SH, 13 μM Cu-yCCS in 50 mM HEPES, pH 7.2, with 100 μM EDTA and 100 μM BCS. After modification with AMS, 2 μg of protein was loaded on 12.5% SDS–PAGE without β-ME. (B) The time course of the disulfide formation with Cu-yCCS or Cu¹⁺-GSH. (▴) Cu-yCCS, (▪) Cu-yCCS with 8 mM GSH/0.16 mM GSSG, (□) Cu¹⁺-GSH, and (▵) Cu¹⁺-GSH with 100 μM BCS. The reaction of 13 μM E,Zn-ySOD1^SH (H48F) with equimolar Cu-yCCS is also shown (•). (C) Time course of the disulfide formation by an equimolar amount of (□) totally reduced apo-yCCS, (▪) oxidized apo-yCCS, and (○) reduced yCCS after reconstitution with Cu¹⁺. (D) Peptide-mapping analysis of oxidized apo-yCCS with and without E,Zn-ySOD1^SH after modification with the thiol-specific reagent, iodoacetamide (IA). After digestion by Asp-N, the sample was purified by ZipTip C18 (Millipore). The peptide fragment, 207–240, containing Cys229 and Cys231 (m/z 3851) is shifted to m/z 3968 after modification with IA. The peptide fragment, 116–152, is also shown in the figure (m/z 3999).

Thiol-disulfide redox potential determination for E,Zn-ySOD1. (A) One of the SDS–PAGE gels for the measurement of the redox equilibrium: 1 μM E,Zn-ySOD1^S–S was mixed with 10 μM GSSG and 0.2–40 mM GSH (from left to right). After anaerobic incubation at 30°C for 65 h, the reaction mixtures were quenched by 10% TCA of total. After modification with AMS, 2 μg of protein was loaded on 12.5% SDS–PAGE under nonreducing conditions. (B) Fraction of the reduced form of ySOD1 was plotted against [GSH]²/[GSSG]. Each plot was fitted with to determine the redox potential, E_SOD1^SH/S–S. Dotted, broken, and solid curves colored red represent the fit for the plot after 14, 38, and 65 h incubation of E,Zn-ySOD1^SH, respectively, while the blue dotted and solid curves after 15 and 65 h incubation of E,Zn-ySOD1^S–S.