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  • 1.
    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)
  • 2.
    Flock, Ulrika
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lachmann, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Watmough, Nicholas J.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase2009In: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 103, no 5, p. 845-850Article in journal (Refereed)
    Abstract [en]

    The c-type nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO+2e(-)+2H(+)-->N(2)O+H(2)O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Adelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O(2) to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O(2) was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.

  • 3.
    Huang, Yafei
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lepp, Håkan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Drici, Nadjia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vectorial proton transfer coupled to reduction of O2 and NO by a heme-copper oxidase2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 51, p. 20257-20262Article in journal (Refereed)
    Abstract [en]

    The heme-copper oxidase (HCuO) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. The HCuOs that reduce O2 to H2O couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. The bacterial NO-reductases (NOR) reduce NO to N2O (2NO + 2e + 2H+ → N2O + H2O), a reaction as exergonic as that with O2. Yet, in NOR both electrons and protons are taken from the outside periplasmic solution, thus not conserving the free energy available. The cbb3-type HCuOs catalyze reduction of both O2 and NO. Here, we have investigated energy conservation in the Rhodobacter sphaeroides cbb3 oxidase during reduction of either O2 or NO. Whereas O2 reduction is coupled to buildup of a substantial electrochemical gradient across the membrane, NO reduction is not. This means that although the cbb3 oxidase has all of the structural elements for uptake of substrate protons from the inside, as well as for proton pumping, during NO reduction no pumping occurs and we suggest a scenario where substrate protons are derived from the outside solution. This would occur by a reversal of the proton pathway normally used for release of pumped protons. The consequences of our results for the general pumping mechanism in all HCuOs are discussed.

  • 4.
    Huang, Yafei
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Singh, Laila M R
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Substrate binding and the catalytic reactions in cbb3-type oxidases: the lipid membrane modulates ligand binding2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, no 6-7, p. 724-31Article in journal (Refereed)
    Abstract [en]

    Heme-copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme-copper oxidases of the cbb3-type are only found in bacteria, often pathogenic ones since they have a low Km for O2, enabling the bacteria to colonize semi-anoxic environments. Cbb3-type (C) oxidases are highly divergent from the mitochondrial-like aa3-type (A) oxidases, and within the heme-copper oxidase family, cbb3 is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N2O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme-copper oxidases are manifested in the lack in cbb3 of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O2 and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O2 and NO reduction as well as the proton-pumping mechanism in all heme-copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein-lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein-membrane interactions in cbb3 oxidases, and possible physiological consequences for this behavior are discussed.

  • 5.
    Lachmann, Peter
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huang, Yafei
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Flock, Ulrika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, p. 25531-25537Article in journal (Refereed)
    Abstract [en]

    Nitric-oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) (2NO + 2H(+) + 2e(-) -> N2O + H2O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe-B. Here, we have studied the reaction between fully reduced NOR and NO using the ""flow-flash"" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of similar to 1 mu s at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to FeB before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 mu M NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.

  • 6.
    Lee, Hyun Ju
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Huang, Yafei
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Functional proton transfer pathways in the heme-copper oxidase superfamily2012In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, no 4, p. 537-544Article, review/survey (Refereed)
    Abstract [en]

    Heme-copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-. B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases.

  • 7.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton transfer in nitric oxide reducing heme-copper oxidases2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Heme-copper oxidases (HCuOs) are best known as terminal oxidases in the aerobic respiratory chain, in which they catalyze the reduction of oxygen to water. By receiving protons and electrons from opposite sides of the membrane as well as pumping protons, HCuOs contribute to the electrochemical proton gradient over the membrane that can be used for ATP synthesis. Divergent members of the HCuO superfamily are nitric oxide reductases (NORs) that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O) as part of the denitrification process, an alternative respiratory pathway.

    The first part of the thesis focuses on electron and proton transfer reactions that are associated with the reductive conversion of NO to N2O and O2 to H2O by the NOR from Paracoccus denitrificans. Our data show that proton uptake in NOR is not electrogenic (protons and electrons are taken up from the same side of the membrane) and that no protons are pumped. Also, structural variants have been investigated and the results suggest a role for these residues in proton transfer. Further, we show that lowering the pH leads to a higher NO reduction rate, while this effect is partially counteracted by a larger degree of substrate inhibition at low pH.

    The second part deals with proton transfer and electrical potential generation in the reaction between the cbb3 oxidase from Rhodobacter sphaeroides and O2 or NO. Our data show that NO reduction by cbb3 oxidase is not coupled to proton translocation and that the direction of proton uptake is dependent on substrate. Our findings suggest that the proton pumping mechanism in HCuOs is incompatible with NO reduction intermediates.

    Finally, experiments on structural variants of the ba3 oxidase from Thermus thermophilus indicate a functional role for the inspected residues in proton transfer and support the suggestion that a single proton-transfer pathway is used in the ba3 oxidase.

  • 8.
    Reimann, Joachim
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Flock, Ulrika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lepp, Håkan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Honigmann, Alf
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A pathway for protons in nitric oxide reductase from Paracoccus denitrificans2007In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1767, no 5, p. 362-373Article in journal (Refereed)
    Abstract [en]

    Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e(-) + 2H(+) -> N2O + H2O) as part of the denitriffication process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O-2-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O-2, we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O-2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O-2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O-2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O-2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

  • 9.
    Smirnova, Irina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chang, Hsin-Yang
    Gennis, Robert B.
    Fee, James A.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Functional Role of Thr-312 and Thr-315 in the Proton-Transfer Pathway in ba3 Cytochrome c Oxidase from Thermus thermophilus2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 33, p. 7033-7039Article in journal (Refereed)
    Abstract [en]

    Cytochrome ba3 from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba3 oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169−16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba3 and O2 in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 → Ser variant in the ba3 oxidase both reactions associated with proton uptake from solution, the PR → F and F → O transitions, were slowed compared to those of wild-type ba3. The observed time constants were slowed 3-fold (for PR → F, from 60 to 170 μs in the wild type) and 30-fold (for F → O, from 1.1 to 40 ms). In the Thr-315 → Val variant, the F → O transition was 5-fold slower (5 ms) than for the wild-type oxidase, whereas the PR → F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical PR → F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O2 reduction in the ba3 oxidase.

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