Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane

doi:10.1073/pnas.2234364100

Aqueous access pathways in subunit a of rotary ATP synthase extend to both sides of the membrane

Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706

Edited by H. Ronald Kaback, University of California, Los Angeles, CA (received for review July 11, 2003)

Abstract

The role of subunit a in promoting proton translocation and rotary motion in the Escherichia coli F₁F_o ATP synthase is poorly understood. In the membrane-bound F_o sector of the enzyme, H⁺ binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 at the center of the membrane via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access pathways have previously been mapped to surfaces of aTMH4. Here we have substituted Cys into the second and fifth TMHs of subunit a and carried out chemical modification with Ag⁺ and N-ethylmaleimide to define the aqueous accessibility of residues along these helices. Access to cAsp-61 at the center of the membrane may be mediated in part by Ag⁺-sensitive residues 248, 249, 251, and 252 in aTMH5. From the periplasmic surface, aqueous access to cAsp-61 may be mediated by silver-sensitive residues 115, 116, 119, 120, 122, and 126 in aTMH2. The Ag⁺-sensitive residues in TMH2, -4, and -5 form a continuum extending from the periplasmic to the cytoplasmic side of the membrane. In an arrangement of helices supported by second-site revertant and crosslinking analyses, these residues cluster at the interior of a four-helix bundle formed by TMH2–5. The aqueous access pathways at the interior of subunit a may be gated by a swiveling of helices in this bundle, alternately exposing cytoplasmic and periplasmic half channels to cAsp-61 during the H⁺ transport cycle.

Footnotes

↵ * To whom correspondence should be addressed at: Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706. E-mail: rhfillin{at}wisc.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ACMA, 9-amino-6-chloro-2-methoxyacridine; NEM, N-ethylmaleimide; TMH, transmembrane helix.
↵ † NIH IMAGE was developed at the National Institutes of Health and is available at http://rsb.info.gov/nih-image.
↵ ‡ We previously reported that the aH245C/D119H mutant membranes, which were made partially functional by the D119H second-site suppressor, were inhibited by ≈50% by 40 μMAg⁺ and nearly completely by 80 μMAg+. The membranes used here were expressed from a plasmid carrying the complete unc operon, vs. the partial operon plasmid used previously, and expressed with an F₁ containing no Cys. These differences likely account for the quite robust activity and slightly reduced Ag+ sensitivity observed here.
↵ § The 14C-NEM labeling of aI248C shown in Fig. 3B appears more extensive than would be predicted from the 38% inhibition shown in Fig. 2B and Table 1. The discrepancy may be due to differences in the conditions used for labeling with 14C-NEM vs. that for inhibition of ATP-driven quenching (see Experimental Procedures).