Mimicking respiratory phosphorylation using purified enzymes

von Ballmoos, Christoph; Biner, Olivier Felix; Nilsson, Tobias; Brzezinski, Peter (2016). Mimicking respiratory phosphorylation using purified enzymes. Biochimica et biophysica acta - bioenergetics, 1857(4), pp. 321-331. Elsevier 10.1016/j.bbabio.2015.12.007

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The enzymes of oxidative phosphorylation are a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.

Item Type:

Journal Article (Original Article)

Division/Institute:

08 Faculty of Science > Departement of Chemistry and Biochemistry

Graduate School:

Graduate School for Cellular and Biomedical Sciences (GCB)

UniBE Contributor:

von Ballmoos, Christoph and Biner, Olivier Felix

Subjects:

500 Science > 570 Life sciences; biology
500 Science > 540 Chemistry

ISSN:

0005-2728

Publisher:

Elsevier

Funders:

[42] Schweizerischer Nationalfonds

Language:

English

Submitter:

Christoph von Ballmoos

Date Deposited:

03 Feb 2016 13:46

Last Modified:

29 May 2017 17:59

Publisher DOI:

10.1016/j.bbabio.2015.12.007

PubMed ID:

26707617

Uncontrolled Keywords:

ATP synthesis; respiratory chain; liposomes; mild uncoupling; ionophore; lateral proton diffusion

BORIS DOI:

10.7892/boris.75009

URI:

https://boris.unibe.ch/id/eprint/75009

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