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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.
    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, ISSN 2041-1723, 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.

  • 3.
    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.

  • 4. 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)
  • 5.
    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)
  • 6.
    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.

  • 7.
    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)
  • 8.
    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)
  • 9. 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)
  • 10. 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)
  • 11. 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)
  • 12. 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.

  • 13. 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)
  • 14. 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.

  • 15. 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, ISSN 1932-6203, 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.

  • 16. 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.

  • 17.
    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).

  • 18. 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).

  • 19. 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)
  • 20. 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)
  • 21. 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, ISSN 2045-2322, 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.

  • 22.
    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, ISSN 2045-2322, 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).

  • 23. 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)
  • 24.
    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.

  • 25.
    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.

  • 26.
    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, ISSN 1932-6203, 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.

  • 27.
    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.

  • 28.
    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)
  • 29. 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.

  • 30.
    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.

  • 31. 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.

  • 32.
    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.

  • 33.
    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.

  • 34. 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, ISSN 2045-2322, 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.

  • 35.
    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).

  • 36.
    Lundin, Camilla Rydström
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Modulation of O-2 reduction in Saccharomyces cerevisiae mitochondria2017In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 24, p. 4049-4055Article in journal (Refereed)
    Abstract [en]

    Respiratory supercomplex factor (Rcf) 1 is a membrane-bound protein that modulates the activity of cytochrome c oxidase (CytcO) in Saccharomycescerevisiae mitochondria. To investigate this regulatory mechanism, we studied the interactions of CytcO with potassium cyanide (KCN) upon removal of Rcf Delta. While the addition of KCN to the wild-type mitochondria results in a full reduction of heme a, with the rcf Delta mitochondria, a significant fraction remains oxidized. Upon addition of ascorbate in the presence of O-2 and KCN, the reduction level of hemes a and b was a factor of similar to 2 larger with the wild-type than with the rcf Delta mitochondria. These data indicate that turnover of CytcO was less blocked in rcf Delta than in the wild-type mitochondria, suggesting that Rcf Delta modulates the structure of the catalytic site.

  • 37.
    Lönneborg, Rosa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Factors that influence the response of the LysR type transcriptional regulators to aromatic compounds2011In: BMC Biochemistry, ISSN 1471-2091, E-ISSN 1471-2091, Vol. 12, p. 49-Article in journal (Refereed)
    Abstract [en]

    Background: The transcriptional regulators DntR, NagR and NtdR have a high sequence identity and belong to the large family of LysR type transcriptional regulators (LTTRs). These three regulators are all involved in regulation of genes identified in pathways for degradation of aromatic compounds. They activate the transcription of these genes in the presence of an inducer, but the inducer specificity profiles are different. Results: The results from this study show that NtdR has the broadest inducer specificity, responding to several nitro-aromatic compounds. Mutational studies of residues that differ between DntR, NagR and NtdR suggest that a number of specific residues are involved in the broader inducer specificity of NtdR when compared to DntR and NagR. The inducer response was also investigated as a function of the experimental conditions and a number of parameters such as the growth media, plasmid arrangement of the LTTR-encoding genes, promoter and gfp reporter gene, and the presence of a His(6) tag were shown to affect the inducer response in E. coli DH5 alpha. Furthermore, the response upon addition of both salicylate and 4-nitrobenzoate to the growth media was larger than the sum of responses upon addition of each of the compounds, which suggests the presence of a secondary binding site, as previously reported for other LTTRs. Conclusions: Optimization of the growth conditions and gene arrangement resulted in improved responses to nitro-aromatic inducers. The data also suggests the presence of a previously unknown secondary binding site in DntR, analogous to that of BenM.

  • 38.
    Lönneborg, Rosa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Smirnova, Irina
    Dian, Cyril
    Leonard, Gordon A
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    In vivo and in vitro investigation of transcriptional regulation by DntR.2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 3, p. 571-82Article in journal (Refereed)
    Abstract [en]

    DntR is a bacterial transcription factor that has been isolated from Burkholderia species that are able to degrade the nitro-aromatic compound 2,4-dinitrotoluene. We recently solved the X-ray crystal structure of DntR, which suggested a putative location of an inducer-binding cavity (IBC). In this study, we constructed mutants of DntR in which residues lining the proposed IBC were modified in order to identify the structural elements involved in inducer binding, to modulate the inducer binding specificity, and to investigate the mechanism of transcriptional regulation by DntR. The transcriptional activation of the reporter gene gfp induced by the wild-type and mutant DntRs was monitored by analysing whole-cell fluorescence using flow-cytometry after addition of a number of potential inducer compounds. Three of the mutant proteins (F111L; F111V/H169V and Y110S/F111V) were purified and the binding constants for several of the potential inducers to these mutants were estimated. Furthermore, crystal structures of the F111L and Y110S/F111V mutant proteins were solved and used to explain changes in the inducer binding specificity at an atomic level. A comparison of the inducing capability in the whole-cell system and binding constants for a number of potential inducers suggests a mechanism where binding of an inducer molecule is not the sole requirement for transcriptional activation. In addition, specific interactions between DntR and the inducer molecule resulting in a conformational change of the protein are needed.

  • 39.
    Lönneborg, Rosa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Varga, Edina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Directed evolution of the transcriptional regulator DntR.: Isolation of mutants with improved DNT responseManuscript (preprint) (Other academic)
  • 40.
    Lönneborg, Rosa
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Varga, Edina
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response.2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 1, p. e29994-(7 pp)Article in journal (Refereed)
    Abstract [en]

    The transcriptional regulator DntR, which previously has been isolated from bacterial strains capable of degrading 2,4-dinitrotoluene (DNT), was engineered in order to improve the ability to detect DNT. A directed evolution strategy was employed, where sequence diversity first was created by random mutagenesis in three subsequent rounds, followed by recombination of previously selected mutants. A gfp gene was used as a reporter for transcriptional activity mediated by DntR and cells with higher GFP expression after addition of DNT were sorted out using fluorescence-activated cell sorting (FACS). A DntR mutant, which displayed 10 times higher induction levels than wild-type DntR in response to DNT was isolated. This mutant still maintained low levels of gfp expression in the absence of DNT. The detection limit was ∼10 µM, a 25-fold improvement compared to wild-type DntR. The functional role of some substitutions found in this clone is discussed in the framework of the structural changes observed when comparing the recently determined structures of DntR with and without bound inducer ligand.

  • 41.
    Namslauer, Andreas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lepp, Håkan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brändén, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jasaitis, Audrius
    Verkhovsky, Michael I.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Plasticity of proton pathway structure and water coordination in cytochrome c oxidase2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 20, p. 15148-15158Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (CytcO) is a redox-driven, membrane-boundproton pump. One of the proton transfer pathways of the enzyme,the D pathway, used for the transfer of both substrate and pumpedprotons, accommodates a network of hydrogen-bonded water moleculesthat span the distance between an aspartate (Asp132), near theprotein surface, and glutamate Glu286, which is an internalproton donor to the catalytic site. To investigate how changesin the environment around Glu286 affect the mechanism of protontransfer through the pathway, we introduced a non-hydrogen-bonding(Ala) or an acidic residue (Asp) at position Ser197 (S197A orS197D), located 7 Å from Glu286. Although Ser197 is hydrogen-bondedto a water molecule that is part of the D pathway "proton wire,"replacement of the Ser by an Ala did not affect the proton transferrate. In contrast, the S197D mutant CytcO displayed a turnoveractivity of 35% of that of the wild-type CytcO, and the O2 reductionreaction was not linked to proton pumping. Instead, a fractionof the substrate protons was taken from the positive ("incorrect")side of the membrane. Furthermore, the pH dependence of theproton transfer rate was altered in the mutant CytcO. The resultsindicate that there is plasticity in the water coordinationof the proton pathway, but alteration of the electrostatic potentialwithin the pathway results in uncoupling of the proton translocationmachinery.

  • 42.
    Namslauer, Ida
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 9, p. 3402-3407Article in journal (Refereed)
    Abstract [en]

    An increasing number of cancer types have been found to be linked to specific mutations in the mitochondrial DNA, which result in specific structural changes of the respiratory enzyme complexes. In this study, we have investigated the effect of 2 such mutations identified in colon cancer patients, leading to the amino acid substitutions Ser458Pro and Gly125Asp in subunit I of cytochrome c oxidase (complex IV) [Greaves et al. (2006) Proc Natl Acad Sci USA 103:714-719]. We introduced these mutations in Rhodobacter sphaeroides, which carries an oxidase that serves as a model of the mitochondrial counterpart. The lack of expression of the former variant indicates that the amino acid substitution results in severely altered overall structure of the enzyme. The latter mutation (Gly171Asp in the bacterial oxidase) resulted in a structurally intact enzyme, but with reduced activity (approximately 30%), mainly due to slowed reduction of the redox site heme a. Furthermore, even though the Gly171Asp CytcO pumps protons, an intrinsic proton leak was identified, which would lead to a decreased overall energy-conversion efficiency of the respiratory chain, and would also perturb transport processes such as protein, ion, and metabolite trafficking. Furthermore, the specific leak may act to alter the balance between the electrical and chemical components of the proton electrochemical gradient.

  • 43.
    Namslauer, Ida
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dietz, Marina
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Functional effects of mutations in cytochrome c oxidase related to prostate cancer2011In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1807, no 10, p. 1336-1341Article in journal (Refereed)
    Abstract [en]

    A number of missense mutations in subunit I of cytochrome c oxidase (CytcO) have previously been linked to prostate cancer (Petros et al. (2005) PNAS, 102, 719). To investigate the effects of these mutations at the molecular level, in the present study we prepared four different structural variants of the bacterial Rhodobacter sphaeroides CytcO (cytochrome aa3), each carrying one amino-acid residue replacement corresponding to the following substitutions identified in the above-mentioned study: Asn11Ser, Ala122Thr, Ala341Ser and Val380Ile (residues Asn25, Ser168, Ala384 and Val423 in the R. sphaeroides oxidase). This bacterial CytcO displays essentially the same structural and functional characteristics as those of the mitochondrial counterpart. We investigated the overall activity, proton pumping and internal electron- and proton- transfer reactions in the structural variants. The results show that the turnover activities of the mutant CytcOs were reduced by at most a factor of two. All variants pumped protons, but in Ser168Thr, Ala384Ser and Val423Ile we observed slight internal proton leaks. In all structural variants the internal electron equilibrium was slightly shifted away from the catalytic site at high pH (10), resulting in a slower observed ferryl to oxidized transition. Even though the effects of the mutations were relatively modest, the results suggest that they destabilize the proton-gating machinery. Such effects could be manifested in the presence of a transmembrane electrochemical gradient resulting in less efficient energy conservation.

  • 44.
    Namslauer, Ida
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lee, Hyun Ju
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A pathogenic mutation in cytochrome c oxidase results in impaired proton pumping while retaining O-2-reduction activity2010In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1797, no 5, p. 550-556Article in journal (Refereed)
    Abstract [en]

    In this work we have investigated the effect of a pathogenic mitochondrial DNA mutation found in human colon cells, at a functional-molecular level. The mutation results in the amino-acid substitution Tyr19His in subunit I of the human CytcO and it is associated with respiratory deficiency. It was introduced into Rhodobacter sphaeroides, which carries a cytochrome c oxidase (cytochrome aa(3)) that serves as a model of the mitochondrial counterpart. The residue is situated in the middle of a pathway that is used to transfer substrate protons as well as protons that are pumped across the membrane. The Tyr33His (equivalent residue in the bacterial CytcO) structural variant of the enzyme was purified and its function was investigated. The results show that in the structurally altered CytcO the activity decreased due to slowed proton transfer; proton transfer from an internal proton donor, the highly-conserved Glu286, to the catalytic site was slowed by a factor of similar to 5, while reprotonation of the Glu from solution was slowed by a factor of similar to 40. In addition, in the structural variant proton pumping was completely impaired. These results are explained in terms of introduction of a barrier for proton transfer through the D pathway and changes in the coordination of water molecules surrounding the Glu286 residue. The study offers an explanation, at the molecular level, to the link between a specific amino-acid substitution and a pathogenic phenotype identified in human colon cells. 

  • 45.
    Nilsson, Tobias
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rydström Lundin, Camilla
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Gustav
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 24113Article in journal (Refereed)
    Abstract [en]

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.

  • 46.
    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.

  • 47.
    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)
  • 48.
    Nordlund, Gustav
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    SNARE-fusion mediated insertion of membrane proteins into native and artificial membranesManuscript (preprint) (Other academic)
  • 49.
    Nordlund, Gustav
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
    SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 4303-Article in journal (Refereed)
    Abstract [en]

    Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

  • 50.
    Nordlund, Gustav
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lönneborg, Rosa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Formation of supported lipid bilayers on silica particles studied using flow cytometry2009In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 25, no 8, p. 4601-4606Article in journal (Refereed)
    Abstract [en]

    Silica colloidal particles with functionalized surfaces are used, for example, in studies of membrane proteins or for drug delivery, where novel applications are based on the use of particles covered by lipid membrane bilayers. The mechanism by which such supported lipid bilayers are formed on spherical support is not fully understood. Here, we present results from studies of this process using a new method based on flow cytometry. The approach enabled us to detect particle populations coated and uncoated with lipids in the same sample according to the vesicle:particle surface area ratio. The data suggest that DOPC lipid vesicles efficiently break upon interaction with the silica colloidal particle surface; only a small fraction of the adsorbed vesicles remain unbroken. Furthermore, the data support earlier observations showing that formation of the lipid bilayer at the surface is a cooperative process, where bilayer formation is catalyzed by previously bound membrane fragments.

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