Sabina Deutschmann

PhD student. U172/U177
sabina.deutschmann@unibe.ch

Importance of lateral enzyme distance on proton coupling of terminal respiratory oxidase and ATP synthase

Background

Respiratory complexes I-IV catalyze the electron transfer from NADH to O2, thereby using the free energy to pump protons out of the cell/cell organelle. Like this, they establish an electrochemical proton gradient called proton motive force (PMF), which is composed of a membrane potential (Δψ) and a transmembrane proton gradient (ΔpH). The PMF acts as an energy source for the ATP synthase. As a last step of oxidative phosphorylation, complex IV couples the reduction of molecular oxygen to water with the generation of a PMF. Following the proton fate after ejection to the P-side, it is interesting to understand its impact on the particular formation of Δψ and ΔpH. In contrast to a substantial contribution to Δψ, a single exported proton will have a minimal effect on ΔpH since it is buffered by the bulk solution, if treated thermodynamically. Especially for alkaliphilic organisms, which lack in ΔpH, this would become a problem as the total pmf is not efficient enough for ATP synthesis. However, it is thought to be a general problem in all organisms. To overcome this problem, experimental and theoretical models suggest a kinetically delayed equilibration of protons with the bulk, which is termed lateral proton transfer along the lipid membrane [1].

Coupled ATP synthesis measurements of bo3oxidase and ATP synthase reconstituted into liposomes (bo3 oxidase acts as PFM generator and the ATP synthase, upon consuming the PMF, synthesized ATP which can be detected) showed effects indicating such an alternative proton fate after ejection to the P-side [2]. Experiments showed that the coupled ATP synthesis activity depends on the lipid composition and on the enzyme density in liposomes. The insertion of negatively charged lipids (DOPG) into basis uncharged lipids (DOPC) led to a drastic decrease in activity. This effect vanishes when the enzyme density in liposomes is diluted below a certain threshold. The membrane composition thus seems to have an effect on proton exchange between the two enzymes. [2] In respect to the above mentioned lateral proton transfer along the membrane surface, the results can suggest that the lipid head groups either influence the distance and rate of protons transferred along the membrane surface, or the average distance of the two enzymes is changed (for schematic model of the latter see Figure 1) [2].

Proton coupling scheme

Figure. 1:
Schematic model showing the effect of negatively charged lipids (DOPG) in uncharged (DOPC) liposomes on the average enzyme distance compared to pure DOPC liposomes, resulting in decreased proton coupling. (From [2])

Project

The aim of the project is to reversibly link the ATP synthase and the bo3 oxidase and to reconstitute the purified complex into liposomes of varying sizes and lipid compositions, followed by coupled ATP synthesis activity measurements. Comparison of ATP synthesis rates before and after cleavage of the crosslink between the two enzymes may shed light on the influence of the average distance of the two enzymes on their proton coupling.

References

  1. Mulkidjanian, Armen Y., Joachim Heberle, and Dmitry A. Cherepanov. “Protons@ interfaces: implications for biological energy conversion.” Biochimica et Biophysica Acta (BBA)-Bioenergetics 1757.8 (2006): 913-930.
  2. Nilsson, Tobias, et al. “Lipid-mediated protein-protein interactions modulate respiration-driven ATP synthesis.” Scientific reports 6 (2016).

Publications

Amati A.M., Graf S., Deutschmann S., Dolder N., von Ballmoos C. (2020) Current problems and future avenues in proteoliposome research. Biochem Soc Trans. 2020 Aug, doi: 10.1042/BST20190966

Previous work in the group:

2014 – 2016 Master student:
Project: The ATP synthase of Vibrio cholerae – the missing link between proton and sodium bioenergetics?