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Reaction of S-cerevisiae mitochondria with ligands: Kinetics of CO and O-2 binding to flavohemoglobin and cytochrome c oxidase
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
Rekke forfattare: 62017 (engelsk)Inngår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1858, nr 2, s. 182-188Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Kinetic methods used to investigate electron and proton transfer within cytochrome c oxidase (CytcO) are often based on the use of light to dissociate small ligands, such as CO, thereby initiating the reaction. Studies of intact mitochondria using these methods require identification of proteins that may bind CO and determination of the ligand-binding kinetics. In the present study we have investigated the kinetics of CO-ligand binding to S. cerevisiae mitochondria and cellular extracts. The data indicate that CO binds to two proteins, CytcO and a (yeast) flavohemoglobin (yHb). The latter has been shown previously to reside in both the cell cytosol and the mitochondrial matrix. Here, we found that yHb resides also in the intermembrane space and binds CO in its reduced state. As observed previously, we found that the yHb population in the mitochondrial matrix binds CO, but only after removal of the inner membrane. The mitochondrial yHb (in both the intermembrane space and the matrix) recombines with CO with T congruent to 270 ms, which is significantly slower than observed with the cytosolic yHb (main component T congruent to 1.3 ms). The data indicate that the yHb populations in the different cell compartments differ in structure.

sted, utgiver, år, opplag, sider
2017. Vol. 1858, nr 2, s. 182-188
Emneord [en]
Electron transfer, Cytochrome aa(3), Yeast, Membrane protein, Ligand, Kinetics, Mechanism
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-140291DOI: 10.1016/j.bbabio.2016.11.009ISI: 000392776400010PubMedID: 27871795OAI: oai:DiVA.org:su-140291DiVA, id: diva2:1081304
Tilgjengelig fra: 2017-03-13 Laget: 2017-03-13 Sist oppdatert: 2019-03-19bibliografisk kontrollert
Inngår i avhandling
1. Modulators of Saccharomyces cerevisiae cytochrome c oxidase: Implications for the regulation of mitochondrial respiration
Åpne denne publikasjonen i ny fane eller vindu >>Modulators of Saccharomyces cerevisiae cytochrome c oxidase: Implications for the regulation of mitochondrial respiration
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Oxidative phosphorylation in mitochondria is performed by enzyme complexes and electron carriers that reside in the inner membrane. It is now generally accepted that these respiratory enzyme complexes assemble into larger so-called supercomplexes. However, it is presently not known why, under which conditions or how these supercomplexes form.

A number of factors of particular importance for the formation of supercomplexes have been identified, such as the Respiratory supercomplex factors (Rcf1 and Rcf2) and cardiolipin. The work presented in this thesis is focused on the characterization of cytochrome c oxidase (CytcO) in mitochondria from Saccharomyces cerevisiae strains in which these components have been removed, with a particular focus on Rcf1. First, we concluded that Rcf1 has an impact on the activity and ligand binding kinetics of CytcO, which upon genetic deletion of rcf1 leads to formation of sub-populations of CytcO with different functionality. Second, we noted that the ability of CytcO to oxidize cytochrome c (cyt. c) depends on the presence of Rcf1. Further, we observed that while CytcO in wild-type mitochondria displayed differences in the oxidation kinetics of cyt. c from horse heart or S. cerevisiae, with the Δrcf1 mitochondria these differences were lost. This observation suggested that Rcf1 interacts with cyt. c. Furthermore, the data showed that in CytcO from Δrcf1 mitochondria heme a3 was altered while heme a was intact.

Using proteo-liposomes of different lipid composition and size we also investigated the influence of lipid head groups on the coupled activity of a quinol oxidase and ATP-synthase. Specifically, we addressed the question if protons are transferred between proton “producers” and “consumers” via lateral proton transfer along the membrane surface or via bulk water. Our data supported the principle of lateral proton transfer.

Lastly, we characterized the ligand binding of yeast flavohemoglobin and concluded that the flavohemoglobin has a population that resides in the intermembrane space of mitochondria, not only in matrix and cytosol as previously suggested.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. s. 48
Emneord
cytochrome c oxidase, cytochrome c, OXPHOS, membrane protein, kinetics, ligand-binding, electron transfer, Rcf1, respiratory supercomplexes, Saccharomyces cerevisiae
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-161515 (URN)978-91-7797-457-4 (ISBN)978-91-7797-456-7 (ISBN)
Disputas
2018-12-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2018-11-20 Laget: 2018-10-29 Sist oppdatert: 2018-11-13bibliografisk kontrollert
2. Regulation of proton-coupled electron transfer in cytochrome c oxidase: The role of membrane potential, proton pathways and ATP
Åpne denne publikasjonen i ny fane eller vindu >>Regulation of proton-coupled electron transfer in cytochrome c oxidase: The role of membrane potential, proton pathways and ATP
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Cytochrome c oxidase (CytcO) is the final electron acceptor of the respiratory chain. In this chain a current of electrons, derived from degradation of nutrients, along with protons, are used to reduce oxygen to water. The reaction is exergonic and the excess energy is used to pump protons across the membrane. This proton-coupled electron transfer is regulated, for example, by the membrane potential, the composition of the membrane and the ATP/ADP concentrations. 

Here, we have investigated the mechanism of this regulation. Specifically, we investigated ligand binding to CytcO in mitochondria, which provides mechanistic information about CytcO in its native environment. In addition to CytcO, a water soluble protein, flavohemoglobin (yHb) was found to bind CO and we found that it is localized in the intermembrane space (IMS). We also extracted CytcO from mitochondria without detergent using the styrene maleic acid (SMA) co-polymer. We could show that the SMA-extracted CytcO behaved similarly in its reaction with O2 and CO as CytcO in mitochondria.

In mitochondria and bacterial membranes CytcO transports charges against a transmembrane electrochemical gradient. We induced a membrane potential across sub-mitochondrial particles (SMPs) by addition of ATP and measured single CytcO turnover. Our results indicate that proton transfer, but not electron transfer, across the membrane is affected by the membrane potential.

In yeast CytcO subunit Cox13 has been shown to play a role in ATP/ADP binding to regulate activity. We have solved the structure of Cox13 using NMR and identified the residues that constitute the ATP-binding site, which is located at the C-terminus.

Finally we showed that the main proton-transfer pathways in yeast CytcO function similarly to their bacterial counterparts and that the proposed H-pathway, absent in bacteria, is not responsible for proton translocation in mitochondrial CytcO from S. cerevisiae.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm university, 2019. s. 53
Emneord
cytochrome c oxidase, charge transfer, membrane potential, membrane protein, mitochondria, ATP, proton pump
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-167130 (URN)978-91-7797-624-0 (ISBN)978-91-7797-625-7 (ISBN)
Disputas
2019-05-10, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, 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 4: Manuscript. Paper 5: Manuscript.

Tilgjengelig fra: 2019-04-15 Laget: 2019-03-19 Sist oppdatert: 2019-03-28bibliografisk kontrollert

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