Cells respond to changes in temperature1,2 or chemical additives3,4 by regulating fatty acid synthesis (FAS) in order to maintain a constant viscosity of their lipid membranes5, suggesting that fundamental processes are dependent on this physical parameter. Understanding the functional basis for characteristic physiochemical properties of cellular structures is a major goal of physical biology, but we often lack the capability to systematically modulate such parameters in vivo. Here we investigate functions for unsaturated lipid composition in Escherichia coli using a metabolic engineering approach, which allows us to titrate inner membrane viscosity across a ten-fold range. We find that unsaturated lipids act as tight regulators of E. coli respiration, an effect that is substrate independent and can be mimicked through heterologous synthesis of branched-chain lipids. We introduce a simple physical model to explain these observations, in which membrane viscosity mediates electron carrier (ubiquinone) collisions and reactivity in the Electron Transport Chain (ETC). Numerical simulations of a diffusion-coupled ETC describe several facets of E. coli respiratory activity, providing a quantitative model for respiration in this model system. We propose that the mobility of ETC components serves as a physical constraint for the evolution of lipid composition in energy-transducing membranes.