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  • 1.
    Berg, Johan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Block, Stephan
    Hook, Fredrik
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Single Proteoliposomes with E.coli Quinol Oxidase: Proton Pumping without Transmembrane Leaks2017In: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, no 5, p. 437-445Article in journal (Refereed)
    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).

  • 2.
    Berg, Johan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liu, Jian
    Svahn, Emelie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ferguson-Miller, Shelagh
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structural changes at the surface of cytochrome c oxidase alter the proton-pumping stoichiometry2020In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1861, no 2, article id 148116Article in journal (Refereed)
    Abstract [en]

    Data from earlier studies showed that minor structural changes at the surface of cytochrome c oxidase, in one of the proton-input pathways (the D pathway), result in dramatically decreased activity and a lower proton-pumping stoichiometry. To further investigate how changes around the D pathway orifice influence functionality of the enzyme, here we modified the nearby C-terminal loop of subunit I of the Rhodobacter sphaeroides cytochrome c oxidase. Removal of 16 residues from this flexible surface loop resulted in a decrease in the proton-pumping stoichiometry to <50% of that of the wild-type enzyme. Replacement of the protonatable residue Glu552, part of the same loop, by an Ala, resulted in a similar decrease in the proton-pumping stoichiometry without loss of the O2-reduction activity or changes in the proton-uptake kinetics. The data show that minor structural changes at the orifice of the D pathway, at a distance of ~40 Å from the proton gate of cytochrome c oxidase, may alter the proton-pumping stoichiometry of the enzyme.

  • 3.
    Berg, Johan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöholm, Johannes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bergstrand, Jan
    Widengren, Jerker
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of cardiolipin in lateral proton transfer along inner mitochondrial membranesManuscript (preprint) (Other academic)
  • 4.
    Björck, Markus L.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Control of transmembrane charge transfer in cytochrome c oxidase by the membrane potential2018In: Nature Communications, E-ISSN 2041-1723, Vol. 9, article id 3187Article in journal (Refereed)
    Abstract [en]

    The respiratory chain in mitochondria is composed of membrane-bound proteins that couple electron transfer to proton translocation across the inner membrane. These charge-transfer reactions are regulated by the proton electrochemical gradient that is generated and maintained by the transmembrane charge transfer. Here, we investigate this feedback mechanism in cytochrome c oxidase in intact inner mitochondrial membranes upon generation of an electrochemical potential by hydrolysis of ATP. The data indicate that a reaction step that involves proton uptake to the catalytic site and presumably proton translocation is impaired by the potential, but electron transfer is not affected. These results define the order of electron and proton-transfer reactions and suggest that the proton pump is regulated by the transmembrane electrochemical gradient through control of internal proton transfer rather than by control of electron transfer.

  • 5.
    Björck, Markus L.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vilhjálmsdóttir, Jóhanna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hartley, Andrew M.
    Meunier, Brigitte
    Näsvik Öjemyr, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marechal, Amandine
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton-transfer pathways in the mitochondrial S. cerevisiae cytochrome c oxidase2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 20207Article in journal (Refereed)
    Abstract [en]

    In cytochrome c oxidase (CytcO) reduction of O-2 to water is linked to uptake of eight protons from the negative side of the membrane: four are substrate protons used to form water and four are pumped across the membrane. In bacterial oxidases, the substrate protons are taken up through the K and the D proton pathways, while the pumped protons are transferred through the D pathway. On the basis of studies with CytcO isolated from bovine heart mitochondria, it was suggested that in mitochondrial CytcOs the pumped protons are transferred though a third proton pathway, the H pathway, rather than through the D pathway. Here, we studied these reactions in S. cerevisiae CytcO, which serves as a model of the mammalian counterpart. We analyzed the effect of mutations in the D (Asn99Asp and Ile67Asn) and H pathways (Ser382Ala and Ser458Ala) and investigated the kinetics of electron and proton transfer during the reaction of the reduced CytcO with O-2. No effects were observed with the H pathway variants while in the D pathway variants the functional effects were similar to those observed with the R. sphaeroides CytcO. The data indicate that the S. cerevisiae CytcO uses the D pathway for proton uptake and presumably also for proton pumping.

  • 6.
    Björck, Markus L.
    et al.
    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.
    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.
    Reaction of S-cerevisiae mitochondria with ligands: Kinetics of CO and O-2 binding to flavohemoglobin and cytochrome c oxidase2017In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1858, no 2, p. 182-188Article in journal (Refereed)
    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.

  • 7. Björklund, Jörgen
    et al.
    Biverståhl, Henrik
    Gräslund, Astrid
    Mäler, Lena
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Real-time transmembrane translocation of penetratin driven by light-generated proton pumping.2006In: Biophys J, ISSN 0006-3495, Vol. 91, no 4, p. L29-31Article in journal (Refereed)
  • 8.
    Brzezinski, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B
    Cytochrome c oxidase: exciting progress and remaining mysteries.2008In: J Bioenerg Biomembr, ISSN 0145-479X, Vol. 40, no 5, p. 521-31Article in journal (Refereed)
  • 9.
    Brzezinski, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 710-23Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c2+ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O2. In this reaction part of the free energy available from O2 reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O2. Our understanding of the molecular design of the machinery that couples O2 reduction to proton pumping in oxidases has greatly benefited from studies of so called "uncoupled" structural variants of the oxidases. In these uncoupled oxidases the catalytic O2-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O2 is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pKa of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pKa presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pKa and uncoupling of proton pumping from O2 reduction.

  • 10.
    Brzezinski, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Moe, Agnes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Structure and Mechanism of Respiratory III-IV Supercomplexes in Bioenergetic Membranes2021In: Chemical Reviews, ISSN 0009-2665, E-ISSN 1520-6890, Vol. 121, no 15, p. 9644-9673Article, review/survey (Refereed)
    Abstract [en]

    In the final steps of energy conservation in aerobic organisms, free energy from electron transfer through the respiratory chain is transduced into a proton electrochemical gradient across a membrane. In mitochondria and many bacteria, reduction of the dioxygen electron acceptor is catalyzed by cytochrome c oxidase (complex IV), which receives electrons from cytochrome bc(1) (complex III), via membrane-bound or watersoluble cytochrome c. These complexes function independently, but in many organisms they associate to form supercomplexes. Here, we review the structural features and the functional significance of the nonobligate III2IV1/2 Saccharomyces cerevisiae mitochondrial super-complex as well as the obligate III2IV2 supercomplex from actinobacteria. The analysis is centered around the Q-cycle of complex III, proton uptake by CytcO, as well as mechanistic and structural solutions to the electronic link between complexes III and IV.

  • 11.
    Brzezinski, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Molecular architecture of the proton diode of cytochrome c oxidase.2008In: Biochem Soc Trans, ISSN 1470-8752, Vol. 36, no Pt 6, p. 1169-74Article in journal (Refereed)
  • 12.
    Brzezinski, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Design principles of proton-pumping haem-copper oxidases.2006In: Curr Opin Struct Biol, ISSN 0959-440X, Vol. 16, no 4, p. 465-72Article in journal (Other academic)
  • 13. Brändén, Gisela
    et al.
    Gennis, Robert B
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Transmembrane proton translocation by cytochrome c oxidase.2006In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1757, no 8, p. 1052-63Article in journal (Refereed)
  • 14. Brändén, Gisela
    et al.
    Pawate, Ashtamurthy S
    Gennis, Robert B
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase.2006In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 103, no 2, p. 317-22Article in journal (Other academic)
  • 15. Brändén, Magnus
    et al.
    Sandén, Tor
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Widengren, Jerker
    Localized proton microcircuits at the biological membrane-water interface.2006In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 103, no 52, p. 19766-70Article in journal (Other academic)
  • 16. Busenlehner, Laura
    et al.
    Brändén, Gisela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Namslauer, Ida
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Armstrong, Richard
    Structural Elements Involved in Proton Translocation by Cytochrome c Oxidase as Revealed by Backbone Amide Hydrogen-Deuterium Exchange of the E286H Mutant2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 1, p. 73-83Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is the terminal electron acceptor in the respiratory chains of aerobic organisms and energetically couples' the reduction of oxygen to water to proton pumping across the membrane. The mechanisms of proton uptake, gating, and pumping have yet to be completely elucidated at the molecular level for these enzymes. For Rhodobacter sphaeroides CytcO (cytochrome aa<sub>3</sub>), it appears as though the E286 side chain of subunit I is a branching point from which protons are shuttled either to the catalytic site for O<sub>2</sub> reduction or to the acceptor site for pumped protons. Amide hydrogen-deuterium exchange mass spectrometry was used to investigate how mutation of this key branching residue to histidine (E286H) affects the structures and dynamics of four redox intermediate states. A functional characterization of this mutant reveals that E286H CytcO retains ∼1% steady-state activity that is uncoupled from proton pumping and that proton transfer from H286 is significantly slowed. Backbone amide H-D exchange kinetics indicates that specific regions of CytcO, perturbed by the E286H mutation, are likely to be involved in proton gating and in the exit pathway for pumped protons. The results indicate that redox-dependent conformational changes around E286 are essential for internal proton transfer. E286H CytcO, however, is incapable of these specific conformational changes and therefore is insensitive to the redox state of the enzyme. These data support a model where the side chain conformation of E286 controls proton translocation in CytcO through its interactions with the proton gate, which directs the flow of protons either to the active site or to the exit pathway. In the E286H mutant, the proton gate does not function properly and the exit channel is unresponsive. These results provide new insight into the structure and mechanism of proton transtocation by CytcO.

  • 17. Busenlehner, Laura S
    et al.
    Salomonsson, Lina
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Armstrong, Richard N
    Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase.2006In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 103, no 42, p. 15398-403Article in journal (Refereed)
  • 18. Chakrabarty, Suman
    et al.
    Namslauer, Ida
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Warshel, Arieh
    Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects2011In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 4, p. 413-426Article in journal (Refereed)
    Abstract [en]

    Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (CcO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent pK(a) values associated with the pH dependencies of specific proton transfer (PT) reactions in CcO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent pK(a) of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated pK(a) values of Glu286 at these different conformations to reproduce the experimentally observed apparent pK(a) of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans CcO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in CcO.

  • 19. Collins, Ruairi
    et al.
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karlberg, Tobias
    Markova, Natalia
    van den Berg, Susanne
    Olesen, Kenneth
    Hammarstrom, Martin
    Flores, Alex
    Schuler, Herwig
    Schiavone, Lovisa Holmberg
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Arner, Elias S. J.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biochemical Discrimination between Selenium and Sulfur 1: A Single Residue Provides Selenium Specificity to Human Selenocysteine Lyase2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 1, p. e30581-Article in journal (Refereed)
    Abstract [en]

    Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

  • 20.
    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, 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.

  • 21.
    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)
  • 22. Devesse, Laurence
    et al.
    Smirnova, Irina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lönneborg, Rosa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kapp, Ulrike
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leonard, Gordon A.
    Dian, Cyril
    Crystal structures of DntR inducer binding domains in complex with salicylate offer insights into the activation of LysR-type transcriptional regulators2011In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 81, no 2, p. 354-367Article in journal (Refereed)
    Abstract [en]

    Activation of LysR-type transcription factors (LTTRs) is thought to result from conformational changes that occur when inducer molecules bind to their Inducer Binding Domains (IBDs). However, the exact nature of these changes remains to be fully elucidated. We present the crystal structures of two truncated constructs of the LTTR DntR in their apo- forms and in complex with its natural inducer molecule, salicylate. These provide a fuller picture of the conformational changes that can occur in LTTR IBDs and offer insights that may be relevant when considering the mechanism of activation of LTTRs. Two of the crystal structures show that DntR IBDs can bind up to two inducer molecules. The full extent of conformational changes observed is achieved only when inducer molecules are bound in both binding sites identified. Point mutations disrupting the putative secondary binding site produce DntR variants with a reduced response to salicylate in a whole cell system, suggesting that this site is functionally relevant.

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  • 23. Di Trani, Justin M.
    et al.
    Gheorghita, Andreea A.
    Turner, Madison
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vahidi, Siavash
    Howell, P. Lynne
    Rubinstein, John L.
    Structure of the bc1cbb3 respiratory supercomplex from Pseudomonas aeruginosa2023In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, no 40, article id e2307093120Article in journal (Refereed)
    Abstract [en]

    Energy conversion by electron transport chains occurs through the sequential transfer of electrons between protein complexes and intermediate electron carriers, creating the proton motive force that enables ATP synthesis and membrane transport. These protein complexes can also form higher order assemblies known as respiratory supercomplexes (SCs). The electron transport chain of the opportunistic pathogen Pseudomonas aeruginosa is closely linked with its ability to invade host tissue, tolerate harsh conditions, and resist antibiotics but is poorly characterized. Here, we determine the structure of a P. aeruginosa SC that forms between the quinol:cytochrome c oxidoreductase (cytochrome bc1) and one of the organism’s terminal oxidases, cytochrome cbb3, which is found only in some bacteria. Remarkably, the SC structure also includes two intermediate electron carriers: a diheme cytochrome c4 and a single heme cytochrome c5. Together, these proteins allow electron transfer from ubiquinol in cytochrome bc1 to oxygen in cytochrome cbb3. We also present evidence that different isoforms of cytochrome cbb3 can participate in formation of this SC without changing the overall SC architecture. Incorporating these different subunit isoforms into the SC would allow the bacterium to adapt to different environmental conditions. Bioinformatic analysis focusing on structural motifs in the SC suggests that cytochrome bc1cbb3 SCs also exist in other bacterial pathogens.

  • 24. Di Trani, Justin M.
    et al.
    Liu, Zhongle
    Whitesell, Luke
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cowen, Leah E.
    Rubinstein, John L.
    Rieske head domain dynamics and indazole-derivative inhibition of Candida albicans complex III2022In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 30, no 1, p. 129-138Article in journal (Refereed)
    Abstract [en]

    Electron transfer between respiratory complexes drives transmembrane proton translocation, which powers ATP synthesis and membrane transport. The homodimeric respiratory complex III (CIII2) oxidizes ubiquinol to ubiquinone, transferring electrons to cytochrome c and translocating protons through a mechanism known as the Q cycle. The Q cycle involves ubiquinol oxidation and ubiquinone reduction at two different sites within each CIII monomer, as well as movement of the head domain of the Rieske subunit. We determined structures of Candida albicans CIII2 by cryoelectron microscopy (cryo-EM), revealing endogenous ubiquinone and visualizing the continuum of Rieske head domain conformations. Analysis of these conformations does not indicate cooperativity in the Rieske head domain position or ligand binding in the two CIIIs of the CIII2 dimer. Cryo-EM with the indazole derivative Inz-5, which inhibits fungal CIII2 and is fungicidal when administered with fungistatic azole drugs, showed that Inz-5 inhibition alters the equilibrium of Rieske head domain positions.

  • 25. Di Trani, Justin M.
    et al.
    Moe, Agnes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Riepl, Daniel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Saura, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kaila, Ville R. I.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rubinstein, John L.
    Structural basis of mammalian complex IV inhibition by steroids2022In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, no 30, article id e2205228119Article in journal (Refereed)
    Abstract [en]

    The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

  • 26. Faxén, Kristina
    et al.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase.2007In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 5, p. 381-386Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).

  • 27.
    Faxén, Kristina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Inside pH Determines Rates of Electron and Proton Transfer in Vesicle-Reconstituted Cytochrome c Oxidase2007In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 5, p. 381-386Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).

  • 28. Faxén, Kristina
    et al.
    Gilderson, Gwen
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    A mechanistic principle for proton pumping by cytochrome c oxidase.2005In: Nature, ISSN 1476-4687, Vol. 437, no 7056, p. 286-9Article in journal (Refereed)
  • 29. Faxén, Kristina
    et al.
    Salomonsson, Lina
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.2006In: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1757, no 5-6, p. 388-94Article in journal (Refereed)
  • 30.
    Fedotovskaya, Olga
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Albertsson, Ingrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Gustav
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hong, Sangjin
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides2021In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1862, no 8, article id 148433Article in journal (Refereed)
    Abstract [en]

    Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc1 and aa3-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O2 reduction activity of the cyt. bc1-aa3 supercomplex. The potential role of the membrane-anchored cyt. cy as a component in supercomplexes was also investigated.

  • 31.
    Fedotovskaya, Olga
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Albertsson, Ingrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Gustav
    Hong, Sangyin
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of a respiratory cytochrome bc1aa3 supercomplex in Rhodobacter sphaeroidesManuscript (preprint) (Other academic)
  • 32. Graf, Simone
    et al.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    The proton pumping bo oxidase from Vitreoscilla2019In: Scientific Reports, E-ISSN 2045-2322, Vol. 9, article id 4766Article in journal (Refereed)
    Abstract [en]

    The cytochrome bo(3) quinol oxidase from Vitreoscilla (vbo(3)) catalyses oxidation of ubiquinol and reduction of O-2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo(3) with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo(3) solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo(3) does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e(-) are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo(3) oxidase of Escherichia coli (ecbo(3)). This coincides with the interpretation that 1 H+/e(-) is pumped across the membrane and 1 H+/e(-) is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo(3) upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo(3) is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo(3) quinol oxidase.

  • 33.
    Graf, Simone
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
    Fedotovskaya, Olga
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kao, Wei-Chun
    Hunte, Carola
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bott, Michael
    von Ballmoos, Christoph
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 34098Article in journal (Refereed)
    Abstract [en]

    Complex III in C. glutamicum has an unusual di-heme cyt.c(1) and it co-purifies with complex IV in a supercomplex. Here, we investigated the kinetics of electron transfer within this supercomplex and in the cyt.aa(3) alone (cyt.bc(1) was removed genetically). In the reaction of the reduced cyt.aa(3) with O-2, we identified the same sequence of events as with other A-type oxidases. However, even though this reaction is associated with proton uptake, no pH dependence was observed in the kinetics. For the cyt. bc(1)-cyt.aa(3) supercomplex, we observed that electrons from the c-hemes were transferred to CuA with time constants 0.1-1 ms. The b-hemes were oxidized with a time constant of 6.5 ms, indicating that this electron transfer is rate-limiting for the overall quinol oxidation/O-2 reduction activity (similar to 210 e(-)/s). Furthermore, electron transfer from externally added cyt.c to cyt.aa(3) was significantly faster upon removal of cyt.bc(1) from the supercomplex, suggesting that one of the c-hemes occupies a position near Cu-A. In conclusion, isolation of the III-IV-supercomplex allowed us to investigate the kinetics of electron transfer from the b-hemes, via the di-heme cyt.c(1) and heme a to the heme a(3)-Cu-B catalytic site of cyt.aa(3).

  • 34. Han, Dan
    et al.
    Namslauer, Andreas
    Pawate, Ashtamurthy
    Morgan, Joel E
    Nagy, Stanislav
    Vakkasoglu, Ahmet S
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B
    Replacing Asn207 by aspartate at the neck of the D channel in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides results in decoupling the proton pump.2006In: Biochemistry, ISSN 0006-2960, Vol. 45, no 47, p. 14064-74Article in journal (Other academic)
  • 35. Hugentobler, Katharina Gloria
    et al.
    Heinrich, Dorothea
    Berg, Johan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Heberle, Joachim
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlesinger, Ramona
    Block, Stephan
    Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase2020In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 21, no 19, article id 6981Article in journal (Refereed)
    Abstract [en]

    The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin. 

  • 36.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hosler, Jonathan P
    Gennis, Robert B
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton Uptake and pK(a) Changes in the Uncoupled Asn139Cys Variant of Cytochrome c Oxidase2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 5, p. 827-836Article in journal (Other academic)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme that links electron transfer from cytochrome c to O-2 to proton pumping across the membrane. Protons are transferred through specific pathways that connect the protein surface with the catalytic site as well as the proton input with the proton output sides. Results from earlier studies have shown that one site within the so-called D proton pathway, Asn139, located similar to 10 angstrom from the protein surface, is particularly sensitive to mutations that uncouple the O-2 reduction reaction from the proton pumping activity. For example, none of the Asn139Asp (charged) or Asn139Thr (neutral) mutant CytcOs pump protons, although the proton-uptake rates are unaffected. Here, we have investigated the Asn139Cys and Asn139Cys/Asp132Asn mutant CytcOs. In contrast to other structural variants investigated to date, the Cys side chain may be either neutral or negatively charged in the experimentally accessible pH range. The data show that the Asn139Cys and Asn139Asp mutations result in the same changes of the kinetic and thermodynamic parameters associated with the proton transfer. The similarity is not due to introduction of charge at position 139, but rather introduction of a protonatable group that modulates the proton connectivity around this position. These results illuminate the mechanism by which CytcO couples electron transfer to proton pumping.

  • 37.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chakrabarty, Suman
    Berthold, Catrine L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Warshel, Arieh
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects2011In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 9, p. 1083-1094Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa(3) CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O(2) reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a pump site and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the HID kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from 02 reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F -> O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P -> F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F -> O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.

  • 38.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Collins, Ruairi
    Arner, Elias S. J.
    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 Discrimination between Selenium and Sulfur 2: Mechanistic Investigation of the Selenium Specificity of Human Selenocysteine Lyase2012In: PLOS ONE, E-ISSN 1932-6203, Vol. 7, no 1, p. e30528-Article in journal (Refereed)
    Abstract [en]

    Selenium is an essential trace element incorporated into selenoproteins as selenocysteine. Selenocysteine (Sec) lyases (SCLs) and cysteine (Cys) desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys, respectively, and generally accept both substrates. Intriguingly, human SCL (hSCL) is specific for Sec even though the only difference between Sec and Cys is a single chalcogen atom. The crystal structure of hSCL was recently determined and gain-of-function protein variants that also could accept Cys as substrate were identified. To obtain mechanistic insight into the chemical basis for its substrate discrimination, we here report time-resolved spectroscopic studies comparing the reactions of the Sec-specific wild-type hSCL and the gain-of-function D146K/H389T variant, when given Cys as a substrate. The data are interpreted in light of other studies of SCL/CD enzymes and offer mechanistic insight into the function of the wild-type enzyme. Based on these results and previously available data we propose a reaction mechanism whereby the Sec over Cys specificity is achieved using a combination of chemical and physico-mechanical control mechanisms.

  • 39.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Role of aspartate 132 at the orifice of a proton pathway in cytochrome c oxidase2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 22, p. 8912-8917Article in journal (Refereed)
    Abstract [en]

    Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e. g., Asn, slows proton uptake by a factor of similar to 5,000. Here, we show that proton uptake at full speed (similar to 10(4) s(-1)) can be restored in the Asp-132-Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139-Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn2+ addition, which in the wildtype cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139-Thr/Asp-132-Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

  • 40.
    Johansson, Ann-Louise
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of acidic residues at the orifice of a proton pathway in cytochrome c oxidaseManuscript (preprint) (Other academic)
  • 41. Kim, Ilsoo
    et al.
    Chakrabarty, Suman
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Warshel, Arieh
    Modeling gating charge and voltage changes in response to charge separation in membrane proteins2014In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, no 31, p. 11353-11358Article in journal (Refereed)
    Abstract [en]

    Measurements of voltage changes in response to charge separation within membrane proteins can offer fundamental information on mechanisms of charge transport and displacement processes. A recent example is provided by studies of cytochrome c oxidase. However, the interpretation of the observed voltage changes in terms of the number of charge equivalents and transfer distances is far from being trivial or unique. Using continuum approaches to describe the voltage generation may involve significant uncertainties and reliable microscopic simulations are not yet available. Here, we attempt to solve this problem by using a coarse-grained model of membrane proteins, which includes an explicit description of the membrane, the electrolytes, and the electrodes. The model evaluates the gating charges and the electrode potentials (c.f. measured voltage) upon charge transfer within the protein. The accuracy of the model is evaluated by a comparison of measured voltage changes associated with electron and proton transfer in bacterial photosynthetic reaction centers to those calculated using our coarse-grained model. The calculations reproduce the experimental observations and thus indicate that the method is of general use. Interestingly, it is found that charge-separation processes with different spatial directions (but the same distance perpendicular to the membrane) can give similar observed voltage changes, which indicates that caution should be exercised when using simplified interpretation of the relationship between charge displacement and voltage changes.

  • 42.
    Król, Sylwia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fedotovskaya, Olga
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, 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.
    Electron and proton transfer in the M. smegmatis III2IV2 supercomplex2022In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1863, no 7, article id 148585Article in journal (Refereed)
    Abstract [en]

    The M. smegmatis respiratory III2IV2 supercomplex consists of a complex III (CIII) dimer flanked on each side by a complex IV (CIV) monomer, electronically connected by a di-heme cyt. cc subunit of CIII. The supercomplex displays a quinol oxidation‑oxygen reduction activity of ~90 e/s. In the current work we have investigated the kinetics of electron and proton transfer upon reaction of the reduced supercomplex with molecular oxygen. The data show that, as with canonical CIV, oxidation of reduced CIV at pH 7 occurs in three resolved components with time constants ~30 μs, 100 μs and 4 ms, associated with the formation of the so-called peroxy (P), ferryl (F) and oxidized (O) intermediates, respectively. Electron transfer from cyt. cc to the primary electron acceptor of CIV, CuA, displays a time constant of ≤100 μs, while re-reduction of cyt. cc by heme b occurs with a time constant of ~4 ms. In contrast to canonical CIV, neither the P → F nor the F → O reactions are pH dependent, but the P → F reaction displays a H/D kinetic isotope effect of ~3. Proton uptake through the D pathway in CIV displays a single time constant of ~4 ms, i.e. a factor of ~40 slower than with canonical CIV. The slowed proton uptake kinetics and absence of pH dependence are attributed to binding of a loop from the QcrB subunit of CIII at the D proton pathway of CIV. Hence, the data suggest that function of CIV is modulated by way of supramolecular interactions with CIII.

  • 43.
    Lee, Hyun Ju
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Svahn, Emelie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Swanson, Jessica M. J.
    Lepp, Hakan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Voth, Gregory A.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Intricate Role of Water in Proton Transport through Cytochrome c Oxidase2010In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 132, no 45, p. 16225-16239Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (CytcO), the final electron acceptor in the respiratory chain, catalyzes the reduction of O-2 to H2O while simultaneously pumping protons across the inner mitochondrial or bacterial membrane to maintain a transmembrane electrochemical gradient that drives, for example, ATP synthesis. In this work mutations that were predicted to alter proton translocation and enzyme activity in preliminary computational studies are characterized with extensive experimental and computational analysis. The mutations were introduced in the D pathway, one of two proton-uptake pathways, in CytcO from Rhodobacter sphaeroides. Serine residues 200 and 201, which are hydrogen-bonded to crystallographically resolved water molecules halfway up the D pathway, were replaced by more bulky hydrophobic residues (Ser200lle, Ser200Val/Ser201Val, and Ser200Val/Ser201Tyr) to query the effects of changing the local structure on enzyme activity as well as proton uptake, release, and intermediate transitions. In addition, the effects of these mutations on internal proton transfer were investigated by blocking proton uptake at the pathway entrance (Asp132Asn replacement in addition to the above-mentioned mutations). Even though the overall activities of all mutant CytcO's were lowered, both the Ser200lle and Ser200Val/Ser201Val variants maintained the ability to pump protons. The lowered activities were shown to be due to slowed oxidation kinetics during the P-R -> F and F -> O transitions (P-R is the "peroxy" intermediate formed at the catalytic site upon reaction of the four-electron-reduced CytcO with O-2, F is the oxoferryl intermediate, and O is the fully oxidized CytcO). Furthermore, the P-R -> F transition is Shown to be essentially pH independent up to pH 12 (i.e., the apparent pK(a) of Glu286 is increased from 9.4 by at least 3 pK(a) units) in the Ser200Val/Ser201Val mutant. Explicit simulations of proton transport in the mutated enzymes revealed that the solvation dynamics can cause intriguing energetic consequences and hence provide mechanistic insights that would never be detected in static structures or simulations of the system with fixed protonation states (i.e., lacking explicit proton transport). The results are discussed in terms of the proton-pumping mechanism of CytcO.

  • 44. Lee, Hyun Ju
    et al.
    Öjemyr, Linda
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vakkasoglu, Ahmet
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B
    Properties of Arg481 mutants of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides suggest that neither R481 nor the nearby D-propionate of heme a3 is likely to be the proton loading site of the proton pump.2009In: Biochemistry, ISSN 1520-4995, Vol. 48, no 30, p. 7123-31Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase utilizes the energy from electron transfer and reduction of oxygen to water and pumps protons across the membrane, generating a proton motive force. A large body of biochemical work has shown that all the pumped protons enter the enzyme through the D-channel, which is apparent in X-ray structures as a chain of water molecules connecting D132 at the cytoplasmic surface of the enzyme to E286, near the enzyme active site. The exit pathway utilized by pumped protons beyond this point and leading to the bacterial periplasm is not known. Also not known is the proton loading site (or sites) which undergoes changes in pKa in response to the chemistry at the enzyme active site and drives the proton pump mechanism. In this paper, we examine the role of R481, a highly conserved arginine that forms an ion pair with the D-propionate of heme a3. The R481H, R481N, R481Q, and R481L mutants were examined. The R481H mutant oxidase is approximately 18% active and pumps protons with approximately 40% of the stoichiometry of the wild type. The R481N, R481Q, and R481L mutants each retain only approximately 5% of the steady-state activity, and this is shown to be due to inhibition of steps in the reaction of O(2) with the reduced enzyme. Neither the R481N mutant nor the R481Q mutant oxidases pump protons, but remarkably, the R481L mutant does pump protons with the same efficiency as the R481H mutant. Since the proton pump is clearly operating in the R481L mutant, these results rule out an essential role in the proton pump mechanism for R481 or its hydrogen bond partner, the D-propionate of heme a3.

  • 45.
    Lepp, Håkan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Internal charge transfer in cytochrome c oxidase at a limited proton supply: proton pumping ceases at high pH.2009In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1790, no 6, p. 552-7Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O(2) is linked to proton uptake from solution to form H(2)O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane. METHODS: In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O(2). These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles. RESULTS: The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O(2) reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH. CONCLUSIONS: The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the "wrong" side of the membrane) and that at this pH the net proton-uptake stoichiometry is approximately 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme. GENERAL SIGNIFICANCE: These results provide new insights into the function of cytochrome c oxidase.

  • 46.
    Lepp, Håkan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Svahn, Emelie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Faxén, Kristina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 17, p. 4929-4935Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase couples electron transfer from cytochrome C to 02 to proton pumping across the membrane. In the initial part of the reaction of the reduced cytochrome c oxidase with 02, an electron is transferred from heme a to the catalytic site, parallel to the membrane surface. Even though this electron transfer is not linked to proton uptake from solution, recently Belevich et al. [(2006) Nature 440, 829] showed that it is linked to transfer of charge perpendicular to the membrane surface (electrogenic reaction). This electrogenic reaction was attributed to internal transfer of a proton from Glu286, in the D proton pathway, to an unidentified protonatable site "above" the heme groups. The proton transfer was proposed to initiate the sequence of events leading to proton pumping. In this study, we have investigated electrogenic reactions in structural variants of cytochrome c oxidase in which residues in the second, K proton pathway of cytochrome c oxidase were modified. The results indicate that the electrogenic reaction linked to electron transfer to the catalytic site originates from charge transfer within the K pathway, which presumably facilitates reduction of the site.

  • 47. Lerche, Michael
    et al.
    Dian, Cyril
    Round, Adam
    Lönneborg, Rosa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leonard, Gordon A.
    The solution configurations of inactive and activated DntR have implications for the sliding dimer mechanism of LysR transcription factors2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 19988Article in journal (Refereed)
    Abstract [en]

    LysR Type Transcriptional Regulators (LTTRs) regulate basic metabolic pathways or virulence gene expression in prokaryotes. Evidence suggests that the activation of LTTRs involves a conformational change from an inactive compact apo-configuration that represses transcription to an active, expanded holo-form that promotes it. However, no LTTR has yet been observed to adopt both configurations. Here, we report the results of structural studies of various forms of the LTTR DntR. Crystal structures of apo-DntR and of a partially autoinducing mutant H169T-DntR suggest that active and inactive DntR maintain a compact homotetrameric configuration. However, Small Angle X-ray Scattering (SAXS) studies on solutions of apo-, H169T- and inducer-bound holo-DntR indicate a different behaviour, suggesting that while apo- DntR maintains a compact configuration in solution both H169T- and holo-DntR adopt an expanded conformation. Models of the SAXS-obtained solution conformations of apo- and holo-DntR homotetramers in complex with promoter-operator region DNA are consistent with previous observations of a shifting of LTTR DNA binding sites upon activation and a consequent relaxation in the bend of the promoter-operator region DNA. Our results thus provide clear evidence at the molecular level which strongly supports the 'sliding dimer' hypothesis concerning LTTR activation mechanisms.

  • 48. Liang, Yingke
    et al.
    Plourde, Alicia
    Bueler, Stephanie A.
    Liu, Jun
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vahidi, Siavash
    Rubinstein, John L.
    Structure of mycobacterial respiratory complex I2023In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, no 13, article id e2214949120Article in journal (Refereed)
    Abstract [en]

    Oxidative phosphorylation, the combined activity of the electron transport chain (ETC) and adenosine triphosphate synthase, has emerged as a valuable target for the treatment of infection by Mycobacterium tuberculosis and other mycobacteria. The mycobacterial ETC is highly branched with multiple dehydrogenases transferring electrons to a membrane-bound pool of menaquinone and multiple oxidases transferring electrons from the pool. The proton-pumping type I nicotinamide adenine dinucleotide (NADH) dehydrogenase (Complex I) is found in low abundance in the plasma membranes of mycobacteria in typical in vitro culture conditions and is often considered dispensable. We found that growth of Mycobacterium smegmatis in carbon-limited conditions greatly increased the abundance of Complex I and allowed isolation of a rotenone-sensitive preparation of the enzyme. Determination of the structure of the complex by cryoEM revealed the “orphan” two-component response regulator protein MSMEG_2064 as a subunit of the assembly. MSMEG_2064 in the complex occupies a site similar to the proposed redox-sensing subunit NDUFA9 in eukaryotic Complex I. An apparent purine nucleoside triphosphate within the NuoG subunit resembles the GTP-derived molybdenum cofactor in homologous formate dehydrogenase enzymes. The membrane region of the complex binds acyl phosphatidylinositol dimannoside, a characteristic three-tailed lipid from the mycobacterial membrane. The structure also shows menaquinone, which is preferentially used over ubiquinone by gram-positive bacteria, in two different positions along the quinone channel, comparable to ubiquinone in other structures and suggesting a conserved quinone binding mechanism. 

  • 49.
    Lobez, Ana Paula
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wu, Fei
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Moe, Agnes
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Electron transfer in the respiratory chain at low salinityManuscript (preprint) (Other academic)
  • 50.
    Lundgren, Camilla A. K
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biner, Olivier
    Bennett, Matthew
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudling, Axel
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinsk, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    von Ballmoos, Christoph
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Scavenging of superoxide by a membrane-bound superoxide oxidase2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, p. 788-793Article in journal (Refereed)
    Abstract [en]

    Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

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