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  • 1.
    Dawitz, Hannah
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schaart, Judith M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Magits, Wout
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rcf1 Modulates Cytochrome c Oxidase Activity Especially Under Energy-Demanding Conditions2020In: Frontiers in Physiology, ISSN 1664-042X, E-ISSN 1664-042X, Vol. 10, article id 1555Article in journal (Refereed)
    Abstract [en]

    The mitochondrial respiratory chain is assembled into supercomplexes. Previously, two respiratory supercomplex-associated proteins, Rcf1 and Rcf2, were identified in Saccharomyces cerevisiae, which were initially suggested to mediate supercomplex formation. Recent evidence suggests that these factors instead are involved in cytochrome c oxidase biogenesis. We demonstrate here that Rcf1 mediates proper function of cytochrome c oxidase, while binding of Rcf2 results in a decrease of cytochrome c oxidase activity. Chemical crosslink experiments demonstrate that the conserved Hig-domain as well as the fungi specific C-terminus of Rcf1 are involved in molecular interactions with the cytochrome c oxidase subunit Cox3. We propose that Rcf1 modulates cytochrome c oxidase activity by direct binding to the oxidase to trigger changes in subunit Cox1, which harbors the catalytic site. Additionally, Rcf1 interaction with cytochrome c oxidase in the supercomplexes increases under respiratory conditions. These observations indicate that Rcf1 could enable the tuning of the respiratory chain depending on metabolic needs or repair damages at the catalytic site.

  • 2.
    Dawitz, Hannah
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schaart, Judith Maria
    Magits, Wout
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rcf1 modulates cytochrome c oxidase activity especially under energy-demanding conditionsManuscript (preprint) (Other academic)
  • 3.
    Nilsson, Tobias
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhou, Shu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Activation of Cytochrome c Oxidase from Saccharomyces cerevisiae by Addition of Respiratory Supercomplex Factor 1Manuscript (preprint) (Other academic)
    Abstract [en]

    In S. cerevisiae the transmembrane protein Respiratory Supercomplex Factor 1 (Rcf1) is involved in formation of the cytochrome c oxidase - bc1 supercomplex. It has also been suggested to mediate electron transfer between the two respiratory enzymes via interactions with cytochrome c. Removal of Rcf1 results in decreased CytcO activity as well as a decrease in the fraction of supercomplexes. The Rcf1 protein can presumably be found as both a monomer and dimer in the membrane. A structure of the latter has been determined using NMR. In this study, we show that co-reconstitution of purified Rcf1 with CytcO from a rcf1Δ strain in liposomes yielded an increase in the CytcO activity. Also, reconstitution of Rcf1 in sub-mitochondrial particles from the rcf1Δ strain yielded an increase in the CytcO activity. However, the increased activity was only observed when the Rcf1 protein was fully unfolded and then refolded in the presence of a membrane. Collectively, the data indicate that Rcf1 can be reconstituted in a membrane as a dimer, but the protein can interact with and reactivate CytcO only in the monomeric form.

  • 4.
    Nitharwal, Ram Gopal
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wiseman, Benjamin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuang, Qie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biochemical and structural characterization of a superoxide dismutase-containing respiratory supercomplex from Mycobacterium smegmatisManuscript (preprint) (Other academic)
  • 5.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wiring Components of the Respiratory Chain: Modulation of the Respiratory Chain in Yeast and Bacteria2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The enzyme complexes of the respiratory chain are organized in supramolecular assemblies, so-called respiratory supercomplexes. In the yeast Saccharomyces cerevisiae, these supercomplexes consist of two copies of complex III (bc1 complex) and one or two copies of complex IV (cytochrome c oxidase, CytcO). Several factors, including lipids and small proteins, have been identified to facilitate or stabilize this organization.

    Respiratory supercomplex factor (Rcf) 1 interacts with CytcO. In this work, we show that in the native S. cerevisiae mitochondrial membrane several forms of CytcO co-exist. Intact CytcO shows spectral and functional properties similar to those of CytcOs from other organisms characterized earlier. A second population displayed a lower midpoint potential of heme a3 as well as accelerated ligand binding, suggesting structural differences around the catalytic site. Severe structural changes of the catalytic site and the overall structure of the enzyme were found in a third population of CytcO. The fraction of the structurally altered CytcO increased upon removal of Rcf1. Here, a mechanism is proposed in which Rcf1 regulates function of the CytcO by altering the catalytic site so that electron transfer between heme a and heme a3 is slowed, resulting in a more exergonic O2-ligand binding. This scenario would in turn increase heat production on the expense of the proton electrochemical gradient.

    Rcf1 was further shown to facilitate electron transfer from the bc1 complex to CytcO in a supercomplex by interacting with the electron carrier cytochrome c (cyt. c).

    In addition, we purified and structurally and functionally characterized the supercomplex of Mycobacterium smegmatis, which contains a membrane-anchored cyt. c as a subunit of the bcc1 complex.

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  • 6.
    Schäfer, Jacob
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dawitz, Hannah
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Regulation of cytochrome c oxidase activity by modulation of the catalytic site2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 11397Article in journal (Refereed)
    Abstract [en]

    The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a3. The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.

  • 7.
    Schäfer, Jacob
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dawitz, Hannah
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ott, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae2018In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, no 9, p. 699-704Article in journal (Refereed)
    Abstract [en]

    Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc1) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O2-reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form.

  • 8.
    Sjöholm, Johannes
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhou, Shu
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rydström Lundin, Camilla
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berg, Johan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Widengren, Jerker
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A membrane-bound anchor for cytochrome c in S. cerevisiaeManuscript (preprint) (Other academic)
  • 9.
    Smirnova, Irina A.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Moscow State University, Russian Federation.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Li, Fei
    Björck, Markus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Östbye, Henrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stanford University, United States.
    von Ballmoos, Christoph
    Lander, Gabriel C.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation of yeast complex IV in native lipid nanodiscs2016In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 12, p. 2984-2992Article in journal (Refereed)
    Abstract [en]

    We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm x 14 nm. Based on this size we estimated that each CytcO was surrounded by similar to 100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondria! membrane, cyt.bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O-2-reduction activity of similar to 130 electrons CytcO(-1) s(-1) and the kinetics of the reaction of the fully reduced CytcO with 02 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

  • 10.
    Wiseman, Benjamin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nitharwal, Ram Gopal
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fedotovskaya, Olga
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schäfer, Jacob
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guo, Hui
    Kuang, Qie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Benlekbir, Samir
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rubinstein, John L.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure of a functional obligate complex III2IV2 respiratory supercomplex from Mycobacterium smegmatis2018In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 25, no 12, p. 1128-1136Article in journal (Refereed)
    Abstract [en]

    In the mycobacterial electron-transport chain, respiratory complex III passes electrons from menaquinol to complex IV, which in turn reduces oxygen, the terminal acceptor. Electron transfer is coupled to transmembrane proton translocation, thus establishing the electrochemical proton gradient that drives ATP synthesis. We isolated, biochemically characterized, and determined the structure of the obligate III2IV2 supercomplex from Mycobacterium smegmatis, a model for Mycobacterium tuberculosis. The supercomplex has quinol:O-2 oxidoreductase activity without exogenous cytochrome c and includes a superoxide dismutase subunit that may detoxify reactive oxygen species produced during respiration. We found menaquinone bound in both the Q(o) and Q(i) sites of complex III. The complex III-intrinsic diheme cytochrome cc subunit, which functionally replaces both cytochrome c(1) and soluble cytochrome c in canonical electron-transport chains, displays two conformations: one in which it provides a direct electronic link to complex IV and another in which it serves as an electrical switch interrupting the connection.

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