Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3 enzymes

Abstract

The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from Escherichia coli, cytochrome bo, for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055-16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P-side of single HCOs. Proton transport activity of cytochrome bo was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH6.4-8.4, while proton release at the P-side had an optimum pH of ~7.4, suggesting that the pH optimum is related to proton release from the proton exit site. Our previous single-enzyme experiments identified rare, long-lived conformation states of cytochrome bo where protons leak back under turn-over conditions. Here, we analyzed and found that ~23% of cytochrome bo proteoliposomes show ΔpH half-lives below 50s after stopping turnover, while only ~5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo exhibit such fast decays. These single-enzyme results confirm our model in which HCO exhibit heterogeneous pumping rates and can adopt rare leak states in which protons are able to rapidly flow back.

A reconstitution method for integral membrane proteins in hybrid lipid-polymer vesicles for enhanced functional durability

“…Only subtle pH changes have been observed in these experiments across all membrane compositions, we interpret that this is due to random orientation of proteins within these vesicle membranes: based on previous reports, this is not unexpected [41]. The pH shifts that we detect are also much smaller than have been observed for cytochrome bo3 at the single vesicle level [42]. New reconstitution strategies will need to be developed in order to enhance and control the orientational bias of reconstituted cytochrome bo3 in hybrid vesicles.…”

Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates

“…to the lateral pressure profile of the lipids) (40,41). Several membrane-bound enzymes have their activity modulated by the intrinsic membrane curvature of the lipid molecules (35)(36)(37)(38)(39)(42)(43)(44). In contrast, the physical curvature of the membrane is simply its shape, independent of its lipid constituents (45).…”

Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase

“…As the proteoliposomes show a notable variation in size and number of (actively) reconstituted enzymes, the ensemble-averaged pH trace is a superposition of several proteoliposome populations and thus depends in a complex way on various parameters. In light of this finding, probing luminal pH values on the level of single liposomes using fluorescence microscopy proved to be a potent approach to analyze the activity of single proton pumps [ 26 , 27 , 28 ]. In this approach, the proteoliposomes are linked to a glass interface and fluorescence microscopy is used to quantify the individual change in fluorescence emission of single, interface-bound proteoliposomes.…”

“…Motivated by these considerations, we set out to investigate the influence of the lipid environment on the proton turnover rate of R. sphaeroides C c O. Inspired by recent studies on a related enzyme [ 26 , 27 , 28 ], this is achieved by reconstituting C c O into liposomes containing a pH sensitive dye and by probing changes of the pH value inside the proteoliposomes (caused by proton uptake by C c O). Analysing pH changes on the level of single proteoliposomes using fluorescence microscopy allows for the determination of single-enzyme kinetics and thus yields information on the proton turnover rate of single C c O in dependence of its lipid environment.…”