Dicarboxylic Acids Link Proton Transfer Across a Liquid Membrane to the Synthesis of Acyl Phosphates. A Model for P-Type H⁺-ATPases

H⁺-ATPases are ion pumps that link proton transfer across cell membranes to the synthesis or hydrolysis of ATP. A current research goal is to understand the molecular-level mechanism of this linking. We present a chemical model that mimics some features of H⁺-ATPases by linking proton transfer across a liquid membrane to the synthesis of acyl phosphates using carboxylic acid anhydride intermediates. Citraconic acid (cis-2-methyl-2-butenedioic acid) accelerated the transfer of protons from a pH 0.3 solution across a chloroform liquid membrane to a pH 10 solution. The mechanism involved spontaneous formation of a small amount of citraconic anhydride (0.6%) in the pH 0.3 layer. This anhydride partitioned into the chloroform layer and diffused to the pH 10 layer, where it hydrolyzed, generating two protons. When the pH 10 layer contained phosphate (1.0 M), some of the citraconic anhydride reacted with phosphate to form citraconyl phosphate, 5.0% yield. In separate experiments, we confirmed that citraconyl phosphate had high phosphoryl donor potential by reacting it with morpholine to form a phosphoramidate (11.5% yield) or with fluoride to form fluorophosphonate (32% yield). To demonstrate the link between an acyl phosphate and a proton gradient in the reverse direction, we used succinyl phosphate, whose hydrolysis occurs in two steps: formation of succinic anhydride, which consumes protons, followed by hydrolysis of succinic anhydride, which releases protons. We generated a pH gradient by carrying out these two steps in separate solutions. Hydrolysis of succinyl phosphate (3.9 mmol) at pH 6.00 started with a increase in pH to 6.16 (0.59 mmol of H⁺ consumed) caused by the formation of succinic anhydride. We extracted this anhydride with dichloromethane and transferred it to a separate solution at pH 6.05. Hydrolysis of the anhydride released protons (0.36 mmol), decreasing the pH to 5.23. Our model suggests that H⁺-ATPases could use acyl phosphates and carboxylic acid anhydride intermediates to link proton transfer to ATP synthesis or hydrolysis.