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 bo₃, 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 bo₃ was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH 6.4–8.4, 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 bo₃ where protons leak back under turn-over conditions. Here, we analyzed and found that ~ 23% of cytochrome bo₃ proteoliposomes show ΔpH half-lives below 50 s after stopping turnover, while only ~ 5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo₃ 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.