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  • 1.
    Ahn, Young O.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Illinois at Urbana-Champaign, USA.
    Albertsson, Ingrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanism of proton transfer through the K-C proton pathway in the Vibrio cholerae cbb(3) terminal oxidase2018Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, nr 11, s. 1191-1198Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The heme-copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O-2 reduction and for proton pumping. This pathway, called the K-C-pathway, starts at Glu-49(P) in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the Kc-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the plc, of an internal proton donor into an experimentally accessible range, from > 10 in wildtype to similar to 8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains similar to 50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.

  • 2. Ahn, Young O.
    et al.
    Lee, Hyun Ju
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kaluka, Daniel
    Yeh, Syun-Ru
    Rousseau, Denis L.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B.
    The two transmembrane helices of CcoP are sufficient for assembly of the cbb(3)-type heme-copper oxygen reductase from Vibrio cholerae2015Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1847, nr 10, s. 1231-1239Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The C-family (cbb(3)) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb(3) from Vibrio cholerae (CcoNOQP(X)) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQP(X) and O-2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c(4), since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the K-C-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

  • 3. Ahn, Young O.
    et al.
    Mahinthichaichan, Paween
    Lee, Hyun Ju
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ouyang, Hanlin
    Kaluka, Daniel
    Yeh, Syun-Ru
    Arjona, Davinia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rousseau, Denis L.
    Tajkhorshid, Emad
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B.
    Conformational coupling between the active site and residues within the K-C-channel of the Vibrio cholerae cbb(3)-type (C-family) oxygen reductase2014Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, nr 42, s. E4419-E4428Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K-C-channel is a conserved glutamate in subunit III. However, the majority of the K-C-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K-C-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb(3) eliminate catalytic activity, but also cause perturbations that propagate over a 28-angstrom distance to the active site heme b(3). The data suggest a linkage between residues lining the KC-channel and the active site of the enzyme, possibly mediated by transmembrane helix alpha 7, which contains both Y241(Vc) and the active site crosslinked Y255(Vc), as well as two Cu-B histidine ligands. Other mutations of residues within or near helix alpha 7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

  • 4.
    Albertsson, Ingrid
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    König, Finja
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, Davinia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ok Ahn, Young
    Gennis, Robert B
    Characterization of the cbb3 terminal oxidase from Helicobacter pyloriManuskript (preprint) (Övrigt vetenskapligt)
  • 5.
    Albertsson, Ingrid
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sjöholm, Johannes
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Royal Institute of Technology (KTH), Sweden.
    ter Beek, Josy
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Watmough, Nicholas J.
    Widengren, Jerker
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans2019Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 9, artikel-id 17234Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Denitrification is a microbial pathway that constitutes an important part of the nitrogen cycle on earth. Denitrifying organisms use nitrate as a terminal electron acceptor and reduce it stepwise to nitrogen gas, a process that produces the toxic nitric oxide (NO) molecule as an intermediate. In this work, we have investigated the possible functional interaction between the enzyme that produces NO; the cd(1) nitrite reductase (cd(1)NiR) and the enzyme that reduces NO; the c-type nitric oxide reductase (cNOR), from the model soil bacterium P. denitrificans. Such an interaction was observed previously between purified components from P. aeruginosa and could help channeling the NO (directly from the site of formation to the side of reduction), in order to protect the cell from this toxic intermediate. We find that electron donation to cNOR is inhibited in the presence of cd(1)NiR, presumably because cd(1)NiR binds cNOR at the same location as the electron donor. We further find that the presence of cNOR influences the dimerization of cd(1)NiR. Overall, although we find no evidence for a high-affinity, constant interaction between the two enzymes, our data supports transient interactions between cd(1)NiR and cNOR that influence enzymatic properties of cNOR and oligomerization properties of cd(1)NiR. We speculate that this could be of particular importance in vivo during metabolic switches between aerobic and denitrifying conditions.

  • 6.
    Arjona, Davinia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wikström, Mårten
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nitric oxide is a potent inhibitor of the cbb(3)-type heme-copper oxidases2015Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 589, nr 11, s. 1214-1218Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    C-type heme-copper oxidases terminate the respiratory chain in many pathogenic bacteria, and will encounter elevated concentrations of NO produced by the immune defense of the host. Thus, a decreased sensitivity to NO in C-type oxidases would increase the survival of these pathogens. Here we have compared the inhibitory effect of NO in C-type oxidases to that in the mitochondrial A-type. We show that O-2-reduction in both the Rhodobacter sphaeroides and Vibrio cholerae C-type oxidases is strongly and reversibly inhibited by submicromolar NO, with an inhibition pattern similar to the A-type. Thus, NO tolerance in pathogens with a C-type terminal oxidase has to rely mainly on other mechanisms.

  • 7. Bhagi-Damodaran, Ambika
    et al.
    Kahle, Maximilian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Shi, Yelu
    Zhang, Yong
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lu, Yi
    Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase2017Ingår i: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 56, nr 23, s. 6622-6626Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Heme-copper oxidase (HCO) is a class of respiratory enzymes that use a heme-copper center to catalyze O-2 reduction to H2O. While heme reduction potential (E degrees') of different HCO types has been found to vary >500 mV, its impact on HCO activity remains poorly understood. Here, we use a set of myoglobin-based functional HCO models to investigate the mechanism by which heme E degrees' modulates oxidase activity. Rapid stopped-flow kinetic measurements show that increasing heme E degrees' by ca. 210 mV results in increases in electron transfer (ET) rates by 30-fold, rate of O-2 binding by 12-fold, O-2 dissociation by 35-fold, while decreasing O-2 affinity by 3-fold. Theoretical calculations reveal that E degrees' modulation has significant implications on electronic charge of both heme iron and O-2, resulting in increased O-2 dissociation and reduced O-2 affinity at high E degrees' values. Overall, this work suggests that fine-tuning E degrees' in HCOs and other heme enzymes can modulate their substrate affinity, ET rate and enzymatic activity.

  • 8.
    Björck, Markus L.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Zhou, Shu
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rydström Lundin, Camilla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reaction of S-cerevisiae mitochondria with ligands: Kinetics of CO and O-2 binding to flavohemoglobin and cytochrome c oxidase2017Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1858, nr 2, s. 182-188Artikel i tidskrift (Refereegranskat)
    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.

  • 9.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase2018Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, nr 11, s. 1223-1234Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Cytochrome c oxidases (CcO) reduce O-2 to H2O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N2O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a true cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N2O can then be formed using only the active-site electrons. In contrast, in CcO, one proton-coupled reduction step most likely has to occur before N2O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme alpha(3)-Cu active site differ with respect to NO reduction efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the rate of NO reduction in CcO.

  • 10.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases2024Ingår i: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 255, artikel-id 112534Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O2 molecule has bound to the diferrous diiron active site and before the O–O bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both N–N bond formation and N–O bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual O–O bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O2 reduction reaction in the family of heme‑copper oxidases.

  • 11.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi. Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reduction of Nitric Oxide to Nitrous Oxide in Flavodiiron Proteins: Catalytic Mechanism and Plausible Intermediates2023Ingår i: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 13, nr 3, s. 2025-2038Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The flavin dependent nonheme diiron proteins comprise a family of enzymes, which can act as scavengers for both molecular oxygen and nitric oxide. The reduction of nitric oxide to nitrous oxide and water in flavodiiron proteins (FDPs) has been studied both experimentally and computationally, but the reaction mechanism is far from well understood. From experiments, it is known that two NO molecules can bind to the reduced active site, forming an observable diferrous dinitrosyl complex. A main question has been whether nitrous oxide can be formed directly from the diferrous dinitrosyl complex or if further reduction and/or protonation is needed to make this step feasible. Experiments have shown that nitrous oxide can be formed in a deflavinated form of the enzyme, indicating that further reduction is not needed. In the present study, hybrid density functional theory calculations are performed on a cluster model of the Thermotoga maritima FDP active site. We show that nitric oxide can be reduced to nitrous oxide and water using a direct coupling mechanism, i.e., without further additions to the reduced active site. The diferrous dinitrosyl complex can form an unstable N-N bridging hyponitrite intermediate, which can rotate into an N-O bond bridging hyponitrite with a low barrier. From this intermediate, the N-O bond cleavage leading to release of nitrous oxide is energetically feasible. An energy profile for the entire catalytic cycle of such a direct coupling mechanism is presented, and it is shown that the suggested mechanism agrees with data on FDP variants. Finally, an energy profile for the entire process starting with the fully reduced enzyme turning over four NO equivalents is constructed. This energy profile suggests explanations to experimentally observed states, such as the dihydroxyl form of the fully oxidized diferric state, and the difference with respect to returning to the original oxidized state after NO reduction between the flavinated and the deflavinated form of the enzyme.

  • 12.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The mechanism for oxygen reduction in cytochrome c dependent nitric oxide reductase (cNOR) as obtained from a combination of theoretical and experimental results2017Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1858, nr 11, s. 884-894Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial NO-reductases (NOR) belong to the heme-copper oxidase (HCuO) superfamily, in which most members are O-2-reducing, proton-pumping enzymes. This study is one in a series aiming to elucidate the reaction mechanisms of the HCuOs, including the mechanisms for cellular energy conservation. One approach towards this goal is to compare the mechanisms for the different types of HCuOs, cytochrome c oxidase (CcO) and NOR, reducing the two substrates O-2 and NO. Specifically in this study, we describe the mechanism for oxygen reduction in cytochrome c dependent NOR (cNOR). Hybrid density functional calculations were performed on large cluster models of the cNOR binuclear active site. Our results are used, together with published experimental information, to construct a free energy profile for the entire catalytic cycle. Although the overall reaction is quite exergonic, we show that during the reduction of molecular oxygen in cNOR, two of the reduction steps are endergonic with high barriers for proton uptake, which is in contrast to oxygen reduction in CcO, where all reduction steps are exergonic. This difference between the two enzymes is suggested to be important for their differing capabilities for energy conservation. An additional result from this study is that at least three of the four reduction steps are initiated by proton transfer to the active site, which is in contrast to CcO, where electrons always arrive before the protons to the active site. The roles of the non-heme metal ion and the redox-active tyrosine in the active site are also discussed.

  • 13.
    Brzezinski, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Moe, Agnes
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structure and Mechanism of Respiratory III-IV Supercomplexes in Bioenergetic Membranes2021Ingår i: Chemical Reviews, ISSN 0009-2665, E-ISSN 1520-6890, Vol. 121, nr 15, s. 9644-9673Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

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

  • 14.
    Brzezinski, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Molecular architecture of the proton diode of cytochrome c oxidase.2008Ingår i: Biochem Soc Trans, ISSN 1470-8752, Vol. 36, nr Pt 6, s. 1169-74Artikel i tidskrift (Refereegranskat)
  • 15.
    Brzezinski, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Design principles of proton-pumping haem-copper oxidases.2006Ingår i: Curr Opin Struct Biol, ISSN 0959-440X, Vol. 16, nr 4, s. 465-72Artikel i tidskrift (Övrigt vetenskapligt)
  • 16.
    Brzezinski, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öjemyr, Linda Näsvik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Intermediates generated during the reaction of reduced Rhodobacter sphaeroides cytochrome c oxidase with dioxygen2013Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1827, nr 7, s. 843-847Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Cytochrome oxidase is one of the functionally most intriguing redox-driven proton pumps. During the last decade our increased understanding of the system has greatly benefited from theoretical calculations and modeling in the framework of three-dimensional structures of cytochrome c oxidases from different species. Because these studies are based on results from experiments, it is important that any ambiguities in the conclusions extracted from these experiments are discussed and elucidated. In a recent study Szundi et al. (Szundi et al. Biochemistry 2012, 51, 9302) investigated the reaction of the reduced Rhodobacter sphaeroides cytochrome c oxidase with O-2 and arrived at conclusions different from those derived from earlier investigations. In this short communication we compare these very recent data to those obtained from earlier studies and discuss the origin of the differences.

  • 17. Di Trani, Justin M.
    et al.
    Gheorghita, Andreea A.
    Turner, Madison
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vahidi, Siavash
    Howell, P. Lynne
    Rubinstein, John L.
    Structure of the bc1cbb3 respiratory supercomplex from Pseudomonas aeruginosa2023Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 120, nr 40, artikel-id e2307093120Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 18. Faxén, Kristina
    et al.
    Gilderson, Gwen
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    A mechanistic principle for proton pumping by cytochrome c oxidase.2005Ingår i: Nature, ISSN 1476-4687, Vol. 437, nr 7056, s. 286-9Artikel i tidskrift (Refereegranskat)
  • 19. Faxén, Kristina
    et al.
    Salomonsson, Lina
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.2006Ingår i: Biochim Biophys Acta, ISSN 0006-3002, Vol. 1757, nr 5-6, s. 388-94Artikel i tidskrift (Refereegranskat)
  • 20.
    Fedotovskaya, Olga
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Albertsson, Ingrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordlund, Gustav
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hong, Sangjin
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides2021Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1862, nr 8, artikel-id 148433Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 21.
    Fedotovskaya, Olga
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Albertsson, Ingrid
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordlund, Gustav
    Hong, Sangyin
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Identification of a respiratory cytochrome bc1aa3 supercomplex in Rhodobacter sphaeroidesManuskript (preprint) (Övrigt vetenskapligt)
  • 22.
    Flock, Ulrika
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lachmann, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Watmough, Nicholas J.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase2009Ingår i: Journal of Inorganic Biochemistry, ISSN 0162-0134, E-ISSN 1873-3344, Vol. 103, nr 5, s. 845-850Artikel i tidskrift (Refereegranskat)
    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.

  • 23.
    Gonska, Nathalie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Young, David R.
    Yuki, Riki
    Okamoto, Takuya
    Antonyuk, Svetlana
    Hasnain, S. Samar
    Muramoto, Kazumasa
    Shiro, Yoshitsugu
    Tosha, Takehiko
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzymeManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Bacterial nitric oxide reductases (NORs) catalyse the reduction of two NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the host. Although NORs are members of the heme-copper oxidase superfamily, and thus relatives of proton-pumping O2-reducing enzymes, the best studied NORs, cNORs (cytochrome c dependent), were found to be non-electrogenic.

    Here, we focus on another type of NOR, qNOR (quinol-dependent). qNOR from Neisseria meningitidis, a human pathogen, was expressed in Escherichia coli and purified as a stable and highly active NO reductase. Spectroscopic and metal analysis of the purified qNOR showed properties largely similar to those in cNORs. Furthermore, the liposome-reconstituted qNOR showed respiratory control ratios consistently above 2, indicative of an electrogenic reaction. We also exchanged residues in a putative proton pathway leading from the cytoplasm to the active site, but there were no significant effects on either turnover rates or electrogenicity. However, the exchange of a glutamate close to the active site (E-498) yielded drastic effects on turnover. We thus suggest that the N. meningitidis qNOR uses cytoplasmic protons, but that the pathway is rather wide and redundant, narrowing around the glutamate-498.

  • 24.
    Gonska, Nathalie
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Young, David
    Yuki, Riki
    Okamoto, Takuya
    Hisano, Tamao
    Antonyuk, Svetlana
    Hasnain, S. Samar
    Muramoto, Kazumasa
    Shiro, Yoshitsugu
    Tosha, Takehiko
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme2018Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 8, artikel-id 3637Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the immune defense of the host. Although NORs belong to the heme-copper oxidase superfamily, comprising proton-pumping O-2-reducing enzymes, the best studied NORs, cNORs (cytochrome c-dependent), are non-electrogenic. Here, we focus on another type of NOR, qNOR (quinol-dependent). Recombinant qNOR from Neisseria meningitidis, a human pathogen, purified from Escherichia coli, showed high catalytic activity and spectroscopic properties largely similar to cNORs. However, in contrast to cNOR, liposome-reconstituted qNOR showed respiratory control ratios above two, indicating that NO reduction by qNOR was electrogenic. Further, we determined a 4.5 angstrom crystal structure of the N. meningitidis qNOR, allowing exploration of a potential proton transfer pathway from the cytoplasm by mutagenesis. Most mutations had little effect on the activity, however the E-498 variants were largely inactive, while the corresponding substitution in cNOR was previously shown not to induce significant effects. We thus suggest that, contrary to cNOR, the N. meningitidis qNOR uses cytoplasmic protons for NO reduction. Our results allow possible routes for protons to be discussed.

  • 25.
    Graf, Simone
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Bern, Switzerland.
    Fedotovskaya, Olga
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kao, Wei-Chun
    Hunte, Carola
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Bott, Michael
    von Ballmoos, Christoph
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum2016Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 6, artikel-id 34098Artikel i tidskrift (Refereegranskat)
    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).

  • 26.
    Huang, Yafei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lepp, Håkan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Drici, Nadjia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Vectorial proton transfer coupled to reduction of O2 and NO by a heme-copper oxidase2008Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, nr 51, s. 20257-20262Artikel i tidskrift (Refereegranskat)
    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.

  • 27.
    Huang, Yafei
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Singh, Laila M R
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Substrate binding and the catalytic reactions in cbb3-type oxidases: the lipid membrane modulates ligand binding2010Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1797, nr 6-7, s. 724-31Artikel i tidskrift (Refereegranskat)
    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.

  • 28.
    Kahle, Maximilian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Appelgren, Sofia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Carroni, Marta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins2023Ingår i: BMC Biology, E-ISSN 1741-7007, Vol. 21, artikel-id 47Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families.

    Results We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples.

    Conclusions Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

  • 29.
    Kahle, Maximilian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Jareck, Sascha
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insights into nitric oxide reductase function from a FeB depleted variantManuskript (preprint) (Övrigt vetenskapligt)
  • 30.
    Kahle, Maximilian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Jareck, Sascha
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Insights into the mechanism of nitric oxide reductase from a Fe-B-depleted variant2019Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 593, nr 12, s. 1351-1359Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (Fe-B). Heme b(3) and Fe-B constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks Fe-B to investigate the role of Fe-B during catalysis. We show that in the Fe-B-less cNOR, binding of both NO and O-2 to heme b(3) still occurs but further reduction is impaired, although to a lesser degree for O-2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

  • 31.
    Kahle, Maximilian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Carroni, Marta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Characterization of the nitric oxide reductase metal insertion chaperones NorQ and NorD from Paracoccus denitrificansManuskript (preprint) (Övrigt vetenskapligt)
  • 32.
    Kahle, Maximilian
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    ter Beek, Josy
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hosler, Jonathan P.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The insertion of the non-heme Fe-B cofactor into nitric oxide reductase from P. denitrificans depends on NorQ and NorD accessory proteins2018Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, nr 10, s. 1051-1058Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial NO reductases (NOR) catalyze the reduction of NO into N2O, either as a step in denitrification or as a detoxification mechanism. cNOR from Paracoccus (P.) denitrificans is expressed from the norCBQDEF operon, but only the NorB and NorC proteins are found in the purified NOR complex. Here, we established a new purification method for the P. denitrificans cNOR via a His-tag using heterologous expression in E. coli. The His-tagged enzyme is both structurally and functionally very similar to non-tagged cNOR. We were also able to express and purify cNOR from the structural genes norCB only, in absence of the accessory genes norQDEF. The produced protein is a stable NorCB complex containing all hemes and it can bind gaseous ligands (CO) to heme b(3), but it is catalytically inactive. We show that this deficient cNOR lacks the nonheme iron cofactor Fe B . Mutational analysis of the nor gene cluster revealed that it is the norQ and norD genes that are essential to form functional cNOR. NorQ belongs to the family of MoxR P-loop AAA + ATPases, which are in general considered to facilitate enzyme activation processes often involving metal insertion. Our data indicates that NorQ and NorD work together in order to facilitate non-heme Fe insertion. This is noteworthy since in many cases Fe cofactor binding occurs spontaneously. We further suggest a model for NorQ/D-facilitated metal insertion into cNOR.

  • 33. Knopp, Michael
    et al.
    Gudmundsdottir, Jonina S.
    Nilsson, Tobias
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    König, Finja
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Warsi, Omar
    Rajer, Fredrika
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Andersson, Dan I.
    De Novo Emergence of Peptides That Confer Antibiotic Resistance2019Ingår i: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, nr 3, artikel-id e00837-19Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from non-coding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes. IMPORTANCE De novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

  • 34.
    Król, Sylwia
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Fedotovskaya, Olga
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Electron and proton transfer in the M. smegmatis III2IV2 supercomplex2022Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1863, nr 7, artikel-id 148585Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 35.
    Lachmann, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Huang, Yafei
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Flock, Ulrika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase2010Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, nr 33, s. 25531-25537Artikel i tidskrift (Refereegranskat)
    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.

  • 36.
    Lee, Hyun Ju
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gennis, Robert B
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Entrance of the proton pathway in cbb3-type heme-copper oxidases2011Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 43, s. 17661-6Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O(2) reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb(3)) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb(3) numbering) in the accessory subunit CcoP (E25(P)). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb(3) by O(2) is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25(P) as the entry point to the proton pathway in cbb(3) and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O(2) reduction. The cbb(3) is in addition to O(2) reduction capable of NO reduction, an activity that was largely retained in the E25(P) variants, consistent with a scenario where NO reduction in cbb(3) uses protons from the periplasmic side of the membrane.

  • 37.
    Moe, Agnes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öjemyr, Linda Näsvik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cryo-EM structure and function of S. pombe complex IV with bound respiratory supercomplex factor2023Ingår i: Communications Chemistry, E-ISSN 2399-3669, Vol. 6, nr 1, artikel-id 32Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Fission yeast Schizosaccharomyces pombe serves as model organism for studying higher eukaryotes. We combined the use of cryo-EM and spectroscopy to investigate the structure and function of affinity purified respiratory complex IV (CIV) from S. pombe. The reaction sequence of the reduced enzyme with O-2 proceeds over a time scale of mu s-ms, similar to that of the mammalian CIV. The cryo-EM structure of CIV revealed eleven subunits as well as a bound hypoxia-induced gene 1 (Hig1) domain of respiratory supercomplex factor 2 (Rcf2). These results suggest that binding of Rcf2 does not require the presence of a CIII-CIV supercomplex, i.e. Rcf2 is a component of CIV. An AlphaFold-Multimer model suggests that the Hig1 domains of both Rcf1 and Rcf2 bind at the same site of CIV suggesting that their binding is mutually exclusive. Furthermore, the differential functional effect of Rcf1 or Rcf2 is presumably caused by interactions of CIV with their different non-Hig1 domain parts. Fission yeast Schizosaccharomyces pombe shares many characteristics with higher eukaryotes. Here, the authors investigate the structure and function of respiratory complex IV from S. pombe, reveal the subunit arrangements and the reaction sequence of O-2 reduction.

  • 38.
    Nilsson, Tobias
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rydström Lundin, Camilla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Nordlund, Gustav
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Ballmoos, Christoph
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Bern, Switzerland.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis2016Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 6, artikel-id 24113Artikel i tidskrift (Refereegranskat)
    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.

  • 39.
    Nilsson, Tobias
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schäfer, Jacob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Zhou, Shu
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Activation of Cytochrome c Oxidase from Saccharomyces cerevisiae by Addition of Respiratory Supercomplex Factor 1Manuskript (preprint) (Övrigt vetenskapligt)
    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.

  • 40.
    Nitharwal, Ram Gopal
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schäfer, Jacob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Wiseman, Benjamin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sjöstrand, Dan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kuang, Qie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biochemical and structural characterization of a superoxide dismutase-containing respiratory supercomplex from Mycobacterium smegmatisManuskript (preprint) (Övrigt vetenskapligt)
  • 41. Petrik, Igor D.
    et al.
    Davydov, Roman
    Kahle, Maximilian
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Sandoval, Braddock
    Dwaraknath, Sudharsan
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Hoffman, Brian
    Lu, Yi
    An Engineered Glutamate in Biosynthetic Models of Heme-Copper Oxidases Drives Complete Product Selectivity by Tuning the Hydrogen-Bonding Network2021Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 60, nr 4, s. 346-355Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome bd oxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O2 binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.

  • 42.
    Poiana, Federica
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Ballmoos, Christoph
    Gonska, Nathalie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Splitting of the O-O bond at the heme-copper catalytic site of respiratory oxidases2017Ingår i: Science Advances, E-ISSN 2375-2548, Vol. 3, nr 6, artikel-id e1700279Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Heme-copper oxidases catalyze the four-electron reduction of O-2 to H2O at a catalytic site that is composed of a heme group, a copper ion (Cu-B), and a tyrosine residue. Results from earlier experimental studies have shown that the O-O bond is cleaved simultaneously with electron transfer from a low-spin heme (heme a/b), forming a ferryl state (P-R; Fe4+= O2-, Cu-B(2+)-OH-). We show that with the Thermus thermophilus ba(3) oxidase, at low temperature (10 degrees C, pH 7), electron transfer from the low-spin heme b to the catalytic site is faster by a factor of similar to 10 (tau congruent to 11 mu s) than the formation of the P-R ferryl (t. 110 ms), which indicates that O-2 is reduced before the splitting of the O-O bond. Application of density functional theory indicates that the electron acceptor at the catalytic site is a high-energy peroxy state [Fe3+-O--O-(H+)], which is formed before the P-R ferryl. The rates of heme b oxidation and P-R ferryl formation were more similar at pH 10, indicating that the formation of the high-energy peroxy state involves proton transfer within the catalytic site, consistent with theory. The combined experimental and theoretical data suggest a general mechanism for O-2 reduction by heme-copper oxidases.

  • 43.
    Reimann, Joachim
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Flock, Ulrika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lepp, Håkan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Honigmann, Alf
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A pathway for protons in nitric oxide reductase from Paracoccus denitrificans2007Ingår i: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1767, nr 5, s. 362-373Artikel i tidskrift (Refereegranskat)
    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.

  • 44.
    Rydström Lundin, Camilla
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Ballmoos, Christoph
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae2016Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, nr 31, s. E4476-E4485Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinolcytochrome c reductase; cyt. bc(1)) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc(1). In the present study, we have investigated the kinetics of ligand binding, the singleturn-over reaction of CytcO with O-2, and the linked cyt. bc(1)-CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1 Delta and rcf2 Delta, respectively). The data show that in the rcf1 Delta and rcf2 Delta strains, in a significant fraction of the population, ligand binding occurs over a time scale that is similar to 100-fold faster (tau congruent to 100 mu s) than observed with the wild-type mitochondria (tau congruent to 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc(1)-CytcO supercomplex. Furthermore, in the rcf1 Delta and rcf2 Delta strains, the single-turnover reaction of CytcO with O-2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electrontransfer bridge from cyt. bc(1) to CytcO via a tightly bound cyt. c. We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc(1) and CytcO.

  • 45. Salomonsson, Lina
    et al.
    Faxén, Kristina
    Ädelroth, Pia
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    The timing of proton migration in membrane-reconstituted cytochrome c oxidase.2005Ingår i: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 102, nr 49, s. 17624-9Artikel i tidskrift (Övrigt vetenskapligt)
  • 46.
    Salomonsson, Lina
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Reimann, Joachim
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tosha, Takehiko
    Krause, Nils
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gonska, Nathalie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Shiro, Yoshitsugu
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Proton transfer in the quinol dependent nitric oxide reductase from geobacillus stearothermophilus during reduction of oxygen2012Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, nr 10, s. 1914-1920Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246]. In this study, we have investigated the reaction between this gNOR and oxygen. Our results show that, like some cNORs, the C. stearothermophilus gNOR is capable of 02 reduction with a turnover of similar to 3 electrons s(-1) at 40 degrees C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) gNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between gNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in gNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

  • 47.
    Schäfer, Jacob
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dawitz, Hannah
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Regulation of cytochrome c oxidase activity by modulation of the catalytic site2018Ingår i: Scientific Reports, E-ISSN 2045-2322, Vol. 8, artikel-id 11397Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 48.
    Schäfer, Jacob
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Dawitz, Hannah
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ott, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structural and functional heterogeneity of cytochrome c oxidase in S. cerevisiae2018Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, nr 9, s. 699-704Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

  • 49.
    Sjöholm, Johannes
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schäfer, Jacob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Zhou, Shu
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Rydström Lundin, Camilla
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Berg, Johan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Widengren, Jerker
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    A membrane-bound anchor for cytochrome c in S. cerevisiaeManuskript (preprint) (Övrigt vetenskapligt)
  • 50.
    Smirnova, Irina A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Moscow State University, Russian Federation.
    Sjöstrand, Dan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Li, Fei
    Björck, Markus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Schäfer, Jacob
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Östbye, Henrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stanford University, United States.
    von Ballmoos, Christoph
    Lander, Gabriel C.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Isolation of yeast complex IV in native lipid nanodiscs2016Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, nr 12, s. 2984-2992Artikel i tidskrift (Refereegranskat)
    Abstract [en]

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

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