Cross-linking of the δ Subunit to One of the Three α Subunits Has No Effect on Functioning, as Expected if δ Is a Part of the Stator That Links the F₁ and F₀ Parts of the Escherichia coli ATP Synthase*

Abstract

A mutant of the Escherichia coliF₁F₀-ATPase has been generated (αQ2C) in which the glutamine at position 2 of the α subunit has been replaced with a cysteine residue. Cu²⁺ treatment of ECF₁from this mutant cross-linked an α subunit to the δ subunit in high yield. Two different sites of disulfide bond formation were involved,i.e. between Cys⁹⁰ (or the closely spaced Cys⁴⁷) of α with Cys¹⁴⁰ of δ, and between Cys² of α and Cys¹⁴⁰ of δ. Small amounts of other cross-linked products, including α-α, δ internal, and α-α-δ were obtained. In ECF₁F₀, there was no cross-linking between the intrinsic Cys of α and Cys¹⁴⁰. Instead, the product generated between Cys² of α and Cys¹⁴⁰ of δ was obtained at near 90% yield. Small amounts of α-α and δ internal were present, and under high Cu²⁺ concentrations, α-α-δ was also formed. The ATPase activity of ECF₁ and ECF₁F₀ was not significantly affected by the presence of these cross-links. When Cys¹⁴⁰ of δ was first modified with N-ethylmaleimide in ECF₁F₀, an α-δ cross-link was still produced, although in lower yield, between Cys⁶⁴ of δ and Cys² of α. ATP hydrolysis-linked proton pumping of inner membranes from the mutant α2QC was only marginally affected by cross-linking of the α to the δ subunit.

These results indicate that Cys¹⁴⁰ and Cys⁶⁴ of the δ subunit and Cys² of the α subunit are in close proximity. This places the δ subunit near the top of the α-β hexagon and not in the stalk region. As fixing the δ to the α by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the δ subunit forms a portion of the stator linking F₁ to F₀.