Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

TL;DR

This study investigates the rotation mechanism of single FoF1-ATP synthases in living bacteria using advanced FRET techniques, providing insights into their in vivo function and enzyme dynamics under physiological conditions.

Contribution

The paper introduces a novel labeling approach for single-molecule FRET, enabling the measurement of subunit rotation of ATP synthase in living bacteria, bridging the gap between in vitro and in vivo analysis.

Findings

Successful measurement of subunit rotation in vivo

New labeling method for single-molecule FRET in bacteria

Insights into enzyme dynamics under physiological proton motive force

Abstract

FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria, chloroplasts and bacteria which provides the 'chemical energy currency' adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP synthesis is driven by a proton motive force (PMF) comprising a proton concentration difference {\Delta}pH plus an electric potential {\Delta}{\Psi} across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within FoF1-ATP synthase is associated with ATP synthesis. Based on intramolecular distance measurements by single-molecule fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation and the step sizes of the different rotor parts have been unraveled. However, these experiments were accomplished in the presence of a PMF consisting of a maximum {\Delta}pH ~ 4 and an unknown {\Delta}{\Psi}. In contrast, in living bacteria the maximum {\Delta}pH across the plasma membrane is likely 0.75, and {\Delta}{\Psi} has been measured between -80 and -140 mV. Thus the problem of in vivo catalytic turnover rates, or the in vivo rotational speed in single FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute number of functional enzymes in a single bacterium required to maintain the high ATP levels has to be determined. We report our progress of measuring subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.