FIGURE 5.

Cross-linking between the HL helix and subunits N and L. A, representative immunoblots of membrane samples from K581C (nuoL) + S290C (nuoN) and L594C (nuoL) + L225C (nuoN) are shown with and without treatment to promote cross-link formation. The disulfide cross-links between HL and N were catalyzed by Cu²⁺ ions. The antibody used was against subunit N. B, representative immunoblot of membrane samples from D546C (nuoL) + D241C (nuoL) is shown with and without treatment to promote cross-link formation. The disulfide cross-link within subunit L was generated by M3M. The antibody against L was used, but as previously noted it did not recognize any cross-linked products. C, the effect of Cu²⁺ ion or MTS reagent (M3M) treatment on the deamino-NADH oxidase activity of the three mutants is shown. Results are the means and S.E. of at least four measurements from at least two membrane preparations. C, proton translocation assays of the three mutants with and without treatment with Cu²⁺ ions or M3M are shown. The fluorescence quenching of ACMA was initiated by deamino-NADH and was reversed by the addition of FCCP.