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J. Biol. Chem., Vol. 282, Issue 12, 9001-9007, March 23, 2007

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Aqueous Access Pathways in ATP Synthase Subunit a

REACTIVITY OF CYSTEINE SUBSTITUTED INTO TRANSMEMBRANE HELICES 1, 3, AND 5^*

From the Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706

Subunit a is thought to play a key role in H⁺ transport-driven rotation of the subunit c ring in Escherichia coli F₁F₀ ATP synthase. In the membrane-traversing F₀ sector of the enzyme, H⁺ binding and release occurs at Asp-61 in the middle of the second transmembrane helix (TMH) of subunit c. Protons are thought to reach Asp-61 via aqueous channels formed at least in part by one or more of the five TMHs of subunit a. Aqueous access to surfaces of TMHs 2, 4, and 5 was previously suggested based upon the chemical reactivity of cysteine residues substituted into these helices. Here we have substituted Cys into TMH1 and TMH3 and extended the substitutions in TMH5 to the cytoplasmic surface. One region of TMH3 proved to be moderately Ag⁺-sensitive and may connect with the Ag⁺-sensitive region found previously on the periplasmic side of TMH2. A single Cys substitution in TMH1 proved to be both N-ethylmaleimide (NEM)-sensitive and Ag⁺-sensitive and suggests a possible packing interaction of TMH1 with TMH2 and TMH3. New Ag⁺- and NEM-sensitive residues were found at the cytoplasmic end of TMH5 and suggest a possible connection of this region to the NEM- and Ag⁺-sensitive region of TMH4 described previously. From the now complete pattern of TMH residue reactivity, we conclude that aqueous access from the periplasmic side of F₀ to cAsp-61 at the center of the membrane is likely to be mediated by residues of TMHs 2, 3, 4, and 5 at the center of a four-helix bundle. Further, aqueous access between cAsp-61 and the cytoplasmic surface is likely to be mediated by residues in TMH4 and TMH5 at the exterior of the four-helix bundle that are in contact with the c-ring.

Received for publication, November 24, 2006 , and in revised form, January 10, 2007.

^* This work was supported by United States Public Health Grant Gm23105 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental table summarizing the properties of all Cys substitutions made in subunit a.

¹ To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253; E-mail: rhfillin{at}wisc.edu.

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