Session: Current Topics in Biological Chemistry:

As the primary producer of adenosine triphosphate (ATP), the energy currency of the cell, F₁F_o ATP synthase is essential for life. F₁F_o is composed of two coupled rotary motors, membrane-embedded F_o and soluble F₁. Using the H⁺ gradient across a membrane, the F_o complex generates torque on the F₁ complex, which synthesizes ATP. During the synthesis process, protons enter F_o through a half channel within subunit a and exit through a half channel at the interface of subunit a (stator) and the c₁₀ ring (rotor). In the reverse process, driven by ATP hydrolysis, protons are pumped in the reverse direction. While much is known about the E. coli complex, which serves as a model for ATP synthase structure and function, aspects of F_o functionality, including the role of some interactions between subunits a and c, remain unresolved. Using site-directed mutagenesis and biochemical assays, we have investigated residues involved in a putative salt bridge across this interface. When Arg50 in subunit c is replaced with Cys, H⁺ movement through F_o ceases; however, the Cys mutant is still able to grow in succinate minimal medium, suggesting that ATP synthesis activity is unaffected. To better understand the necessary chemical properties of residues at these positions, we report the results of H⁺ permeability, H⁺ pumping, and ATP synthesis experiments showing the remarkable tolerance of these regions to mutation (with the notable exception of Cys) and the effects of mutations on H⁺ movement in the ATP-hydrolysis vs. ATP-synthesis directions.

Ryan Steed

Assistant Professor, University of North Carolina Asheville

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