Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing “molecular bellows” model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly.

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