Abstract
Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100–200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.
Highlights
Adenosine triphosphate (ATP) is the universal cellular energy currency
oxidative phosphorylation (OXPHOS) enzymes are in cell membrane patches extending several tens of nm
E. coli strains were engineered by ‘scarlessly’ replacing the native allele of interest on the genome with genes for fluorescently labeled proteins — GFP or mCherry fused with subunits of one of five OXPHOS enzymes
Summary
Adenosine triphosphate (ATP) is the universal cellular energy currency. The energy released upon hydrolysis of ATP powers many biological processes. Most organisms meet ATP demands by oxidative phosphorylation (OXPHOS), a multi-enzyme process spanning all domains of life using nutrient molecule catabolism. In OXPHOS, electrontransfer reactions between membrane-associated enzymes are coupled via the proton-motive force (pmf) to ATP synthesis by FoF1ATPase [1,2]. Enzymes have been studied extensively for mitochondrial OXPHOS in terms of structure/biochemistry [2], with significant understanding of gene expression/regulation stemming from prokaryotes [3]. The system-level architecture and dynamics of OXPHOS membranes are not resolved
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