Abstract
In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring a c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. The simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.
Highlights
FoF1-ATP synthase (FoF1) is a ubiquitous enzyme that synthesizes or hydrolyzes ATP coupled with proton translocation at the inner mitochondrial membrane, chloroplast thylakoid membrane, and bacterial plasma membrane (Boyer, 1997; Walker, 2013; Yoshida et al, 2001)
Schematic picture of the a-subunit and the analysis of the simulation trajectories, we found that prolonged duration times for proton uptake in the two mutated c-subunits can be shared
We previously produced a fusion mutant, c10 FoF1, in which 10 copies of the Fo-c subunit in the c10-ring were fused into a single polypeptide, and demonstrated that c10 FoF1 was active in proton-coupled ATP hydrolysis/synthesis (Mitome et al, 2004)
Summary
FoF1-ATP synthase (FoF1) is a ubiquitous enzyme that synthesizes or hydrolyzes ATP coupled with proton translocation at the inner mitochondrial membrane, chloroplast thylakoid membrane, and bacterial plasma membrane (Boyer, 1997; Walker, 2013; Yoshida et al, 2001). FoF1 synthesizes ATP via rotation of the central rotor driven by the proton motive force across the membrane. The enzyme comprises two rotary motors that share the rotor, that is, the water soluble F1, which has catalytic sites for ATP synthesis/hydrolysis (Noji et al, 2017), and the membrane-e mbedded Fo, which mediates proton translocation (Kühlbrandt, 2019). The Fo motor consists of a c oligomer ring (c-ring), which serves as the rotor, and the ab stator portion located on the c-ring periphery. Downgradient proton translocation through Fo drives rotation of the central rotor composed of a c-ring and γε subunits, Mitome, Kubo, et al eLife 2022;10:e69096.
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