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Single Proteoliposomes with E.coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Rekke forfattare: 42017 (engelsk)Inngår i: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, nr 5, s. 437-445Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Respiratory oxidases are transmembrane enzymes that catalyze the reduction of dioxygen to water in the final step of aerobic respiration. This process is linked to proton pumping across the membrane. Here, we developed a method to study the catalytic turnover of the quinol oxidase, cytochromebo(3) from E.coli at single-molecule level. Liposomes with reconstituted cytochromebo(3) were loaded with a pH-sensitive dye and changes in the dye fluorescence, associated with proton transfer and pumping, were monitored as a function of time. The single-molecule approach allowed us to determine the orientation of cytochromebo(3) in the membrane; in approximate to 70% of the protein-containing liposomes protons were released to the outside. Upon addition of substrate we observed the buildup of a pH (in the presence of the K+ ionophore valinomycin), which was stable over at least approximate to 800s. No rapid changes in pH (proton leaks) were observed during steady state proton pumping, which indicates that the free energy stored in the electrochemical gradient in E.coli is not dissipated or regulated through stochastic transmembrane proton leaks, as suggested from an earlier study (Li etal. J. Am. Chem. Soc. (2015) 137, 16055-16063).

sted, utgiver, år, opplag, sider
2017. Vol. 57, nr 5, s. 437-445
Emneord [en]
proton translocation, single molecule, proton pump, electron transfer, membrane protein
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-144709DOI: 10.1002/ijch.201600138ISI: 000401329000010OAI: oai:DiVA.org:su-144709DiVA, id: diva2:1127988
Tilgjengelig fra: 2017-07-20 Laget: 2017-07-20 Sist oppdatert: 2020-01-07bibliografisk kontrollert
Inngår i avhandling
1. Proton transfer across and along biological membranes
Åpne denne publikasjonen i ny fane eller vindu >>Proton transfer across and along biological membranes
2020 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Proton-transfer reactions belong to the most prevalent reactions in the biosphere and make life on Earth possible, as they are central to energy conversion. In most known organisms, protons are translocated from one side of a membrane to the other, which generates an electrochemical gradient that drives ATP synthesis. Both the membranes and the proteins that are involved in these processes are vital components of energy-conversion machineries. This thesis presents and discusses proton transfer at surfaces of membranes and proteins, as well as proton translocation across membranes via enzymes.

In the first work, we developed a single-enzyme approach to study proton translocation by the proton pump cytochrome bo3 (cyt. bo3). The generated proton gradients were stable as long as substrate (electrons, oxygen) was available. Individual cyt. bo3 could generate proton gradients of ∼2 pH units, which correspond to the measured electrochemical gradient in Escherichia coli cells.

When acidic and basic amino acids are in close proximity to each other on a protein surface, their individual Coulomb cages can merge to form a proton antenna that enables fast proton transfer to specific groups. To investigate how the function of a proton pump is affected by structural changes in a proton antenna, close to a proton uptake pathway, we characterized the function and structure of genetic variants of cytochrome c oxidase (CytcO). When a Glu, located about 10 Å from the first residue of the D-pathway, was replaced by a non-protonatable residue (Ala) the proton pumping efficiency decreased by more than half compared to the wild-type enzyme. The proton-uptake kinetics was also altered in this variant.

Cardiolipin (CL) is found in membranes where ATP is generated. This phospholipid alters the membrane structure and binds a variety of proteins including all complexes that take part in oxidative phosphorylation. To investigate the role of CL in proton-transfer reactions on the surface of membranes we used fluorescence correlation spectroscopy to study inner mitochondrial membranes from Saccharomyces cerevisiae. The protonation rate at wild-type membranes was about 50% of that measured with membranes prepared from mitochondria lacking CL. The protonation rate on the surface of small unilamellar vesicles (SUVs) decreased by about a factor of three when DOPC-SUVs were supplemented with 20% CL. Furthermore, phosphate buffer titrations with SUVs showed that CL can act as a local proton buffer in a membrane.

The respiratory supercomplex factor 1 (Rcf1) has been suggested to facilitate direct electron transfer from the bc1 complex to CytcO by bridging the enzymes and binding cytochrome c (cyt. c) to a flexible domain of Rcf1. We investigated biding of cyt. c to Rcf1 reconstituted into different membrane environments. The apparent KD of the binding between cyt. c and DOPC-liposomes was almost five times lower when Rcf1 was present in the vesicles. Moreover, the apparent KD between cyt. c and liposome reconstituted CytcO was about nine times lower for CytcO isolated from a wild-type strain compared to a Rcf1-lacking strain.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. s. 93
Emneord
biological membranes, cardiolipin, cytochrome bo3, cytochrome c oxidase, energy conversion, fluorescence correlation spectroscopy (FCS), localized coupling, mitochondria, proton transfer, Rcf1, respiration, single-enzyme measurement
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-177422 (URN)978-91-7797-941-8 (ISBN)978-91-7797-942-5 (ISBN)
Disputas
2020-02-21, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Tilgjengelig fra: 2020-01-29 Laget: 2020-01-06 Sist oppdatert: 2020-01-23bibliografisk kontrollert

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