Chemical reactivities of cysteine substitutions in subunit a of ATP synthase define residues gating H+ transport from each side of the membrane

Abstract

Subunit a plays a key role in coupling H(+) transport to rotations of the subunit c-ring in F(1)F(o) ATP synthase. In Escherichia coli, H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of F(o) subunit c. Based upon the Ag(+) sensitivity of Cys substituted into subunit a, H(+) are thought to reach Asp-61 via aqueous pathways mapping to surfaces of TMH 2-5. In this study we have extended characterization of the most Ag(+)-sensitive residues in subunit a with cysteine reactive methanethiosulfonate (MTS) reagents and Cd(2+). The effect of these reagents on ATPase-coupled H(+) transport was measured using inside-out membrane vesicles. Cd(2+) inhibited the activity of all Ag(+)-sensitive Cys on the cytoplasmic side of the TMHs, and three of these substitutions were also sensitive to inhibition by MTS reagents. On the other hand, Cd(2+) did not inhibit the activities of substitutions at residues 119 and 120 on the periplasmic side of TMH2, and residues 214 and 215 in TMH4 and 252 in TMH5 at the center of the membrane. When inside-out membrane vesicles from each of these substitutions were sonicated during Cd(2+) treatment to expose the periplasmic surface, the ATPase-coupled H(+) transport activity was strongly inhibited. The periplasmic access to N214C and Q252C, and their positioning in the protein at the a-c interface, is consistent with previous proposals that these residues may be involved in gating H(+) access from the periplasmic half-channel to Asp-61 during the protonation step.

FIGURE 1.

Location of the most Ag⁺-sensitive cysteine substitutions in a topological model of subunit a. The ATP-driven proton pumping activity of the Cys substitutions highlighted in red was inhibited by >90% on treatment with 40 μm Ag⁺. Residues colored in gray were less sensitive to inhibition by Ag⁺. Numbered residues that are shaded in light gray define the positions of disulfide cross-link formation in doubly Cys-substituted mutants. The position of the essential Arg-210 residue in TMH4 is indicated.

FIGURE 2.

Cross-sectional view of positions of Ag⁺-sensitive residues in transmembrane regions of subunit a. Cys substitutions showing >90% inhibition of ATP-driven ACMA quenching on treatment with 40 μm Ag⁺ are depicted in red. The orientation of TMHs 2–4 in a four-helix bundle is suggested by disulfide cross-link formation between the residues connected by fine solid lines. The green shaded areas depict the cross-linkable faces of aTMH4 with cTMH2. TMHs 4 and 5 are predicted to swivel counterclockwise and clockwise, respectively, during the gating of proton access to cAsp-61 from the periplasmic half-channel in the center of the four-helix bundle.

FIGURE 3.

Inhibition of ATP-driven proton pumping by NEM, MTSM, and MTSET with membrane vesicles from the S202C, S206C, and N214C substitutions. ATP-driven quenching of ACMA fluorescence by inverted membrane vesicles was assayed as described under “Experimental Procedures” using HMK-nitrate buffer. Fluorescence quenching was initiated by the addition of ATP at 20 s and terminated by the addition of nigericin at 100 s. The return to the maximum fluorescence reached after the addition of nigericin was used to calculate the relative inhibition of quenching. Fluorescence quenching by mutant vesicles treated with DMSO (control), 5 mm NEM, 2 mm MTSM, and 2 mm MTSEA.

FIGURE 4.

ATP-driven proton pumping activity with different substitutions in Ser-202 of subunit a. The activity of the S202A, S202I, S202M, and S202K substitutions are compared with wild type.

FIGURE 5.

Inhibition of ATP-driven proton pumping by Cd²⁺ with S206C-substituted subunit a. ATP-driven quenching of ACMA fluorescence was measured as described under “Experimental Procedures” using HMK-chloride buffer. Membrane vesicles were treated with varying concentrations of CdCl₂ for 10 min at room temperature prior to initiation of the assay with ATP. The return of the fluorescent signal to the maximum reached after the addition of nigericin was used to calculate the inhibition values given in Fig. 6. The activity and extent of Cd²⁺ inhibition of wild-type (A) and S206C (B) membrane vesicles are compared.

FIGURE 6.

Concentration dependence of Cd²⁺ inhibition of ATP-driven proton pumping with different Cys substitutions in subunit a. The percent inhibition of ATP-driven proton pumping was calculated as described in Fig. 5.

FIGURE 7.

Relative inhibition of ATP-driven proton pumping by Cd²⁺ with Cys substitutions in TMHs 2, 4, and 5 of subunit a. Membrane vesicles were treated with 1 mm Cd²⁺ and the extent of inhibition calculated as described in Fig. 5. The position of the substituted residues in a two dimensional model of subunit a is indicated in Fig. 1.

FIGURE 8.

Effect of sonication during Cd²⁺ treatment on inhibition of ATP-driven proton pumping with L120C membrane vesicles. Inverted membrane vesicles were treated or not treated with 1 mm Cd²⁺ and subjected or not subjected to 1 min of sonication as described under “Experimental Procedures.”

FIGURE 9.

Effect of sonication during Cd²⁺ treatment on inhibition of ATP-driven proton pumping with M215C and Q252C membrane vesicles. Inverted membrane vesicles were treated with 1 mm Cd²⁺ and subjected to 1 min of sonication as described under “Experimental Procedures.” Control membrane vesicles were sonicated in the absence of Cd²⁺. A, wild type; B, M215C; C, Q252C.

FIGURE 10.

Sonication potentiates Cd²⁺ inhibition with Cys substitutions that may be aqueous inaccessible from the cytoplasmic side of inverted membrane vesicles. Experiments were carried out as described in Figs. 8 and 9 and relative inhibition calculated as described in the legend to Fig. 5. Open bars, no sonication; grey bars, with sonication.

FIGURE 11.

Sonication potentiates Cd²⁺ inhibition with the H245C substitution in aTMH5. Inverted membrane vesicles were treated with 1 mm Cd²⁺ and subjected or not subjected to 1 min of sonication as described in Fig. 8. A, quenching response of vesicles sonicated in the presence or absence of Cd²⁺. B, quenching response of non-sonicated vesicles in the presence or absence of Cd²⁺. The likely location of H245C on the periplasmic face at the center of the four-helix bundle is indicated in Figs. 1 and 2.