Single Proton Pump Activity Measurements on Single Vesicles for a Quinol Heme-Copper Oxidase

Heme-copper oxidases are key elements in the respiratory chain pumping protons cellular membrane (1, 2) and generate the electrical and chemical potential that drives ATP synthesis. Understanding the precise proton pumping mechanism has been a central theme in bionergetics research over the last decades (1, 2). However crucial mechanistic details underlying heme-copper oxidase function and regulation remain masked in conventional techniques due to ensemble averaging. Here we have developed a novel method to monitor for the first time with single molecule resolution the proton pumping activity of the quinol heme-copper oxidase, cytochrome bo3 reconstituted in liposomes. using our recently developed arrays of surface tethered liposomes (3-5) and coupling electrochemistry with fluorescent microscopy allowed us for the first time to in situ activate and simultaneously monitor bo3pumping activity in liposomes loaded with pH sensitive fluorescent reporters. Imaging in a massively parallel manner (103-104 liposomes) and at the single enzyme level allowed to directly observe, quantify the activity rates, abundance and lifetimes, of a plethora of interconverting long-lived (min) functional states. Parallel and multiplexed single vesicle and single molecule readout gave access to the full distribution of rates across the ensemble of proton pumps, and importantly how pH and membrane regulatory inputs modulate the average as well as the full fluctuation spectrum of bo3 proton pump. References 1. K. Faxen, G. Gilderson, P. Adelroth, P. Brzezinski, Nature437, 286, 2005. 2. I. Belevich, M. I. Verkhovsky, M. Wikstrom, Nature440, 829, 2006. 3. N. S. Hatzakis et al., Nat. Chem. Biol.5, 835, 2009. 4. S. M. Christensen, P.-Y. Bolinger, N. S. Hatzakis, M. W. Mortensen, D. Stamou, Nat. Nanotech.7, 51, 2011. 5. N. S. Hatzakis et al., J. Am. Chem. Soc.134, 9296, 2012.