| 1. |
- Ahn, Young O., et al.
(författare)
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Conformational coupling between the active site and residues within the K-C-channel of the Vibrio cholerae cbb(3)-type (C-family) oxygen reductase
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 111:42, s. E4419-E4428
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Ahn, Young O., et al.
(författare)
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Mechanism of proton transfer through the K-C proton pathway in the Vibrio cholerae cbb(3) terminal oxidase
- 2018
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1859:11, s. 1191-1198
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Ahn, Young O., et al.
(författare)
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The two transmembrane helices of CcoP are sufficient for assembly of the cbb(3)-type heme-copper oxygen reductase from Vibrio cholerae
- 2015
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1847:10, s. 1231-1239
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
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| 5. |
- Albertsson, Ingrid, et al.
(författare)
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Functional interactions between nitrite reductase and nitric oxide reductase from Paracoccus denitrificans
- 2019
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Albertsson, Ingrid, 1984-
(författare)
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Heme-copper oxidases and nitric oxide : Reaction mechanisms and supercomplex formation
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the denitrification process where nitrate is stepwise reduced to nitrogen gas, the toxic molecule nitric oxide (NO) is formed as an intermediate. Nitric oxide is produced by the enzyme cd1 Nitrite reductase (cd1NiR) by reduction of nitrite and is later reduced to nitrous oxide by nitric oxide reductase (cNOR).We have investigated if a complex, with the role to facilitate rapid removal of NO, is formed between the two enzymes cd1NiR and cNOR in the bacterium P. denitrificans. Using activity measurements, we found transient interactions between the two enzymes influencing enzymatic activity of cNOR and dimerization of cd1NiR. This complex formation may be important in the transition between aerobic and anaerobic respiration of the bacteria.Heme-copper oxidases are terminal components of the respiratory chain, catalysing the reduction of oxygen to water. We have investigated possible supercomplex formation between the bc1-complex and the heme-copper oxidase of either the aa3 or cbb3 type, in the respiratory chain of Rhodobacter sphaeroides. We found evidence for a functional supercomplex between bc1-aa3, but not between bc1-cbb3.The C-type (cbb3) oxidase from Vibrio cholerae has one proton transfer pathway for delivering protons both to the catalytic site and for protons being pumped. We identified an internal proton donor (XH), which could act as a branching point in the pathway. We also suggest which residues may function as this branching point; Y321 or Y321 and N293 together with water molecules. We were also interested in the C-type oxidase from Helicobacter pylori and its interaction with NO. We successfully expressed and purified an active H. pylori cbb3 in V. cholerae. We found that this cbb3 was reversibly inhibited by nitric oxide similar to other oxidases, and that it also displayed a nitric oxide reductase activity.
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| 7. |
- Arjona, Davinia, et al.
(författare)
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Nitric oxide is a potent inhibitor of the cbb(3)-type heme-copper oxidases
- 2015
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 589:11, s. 1214-1218
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Bhagi-Damodaran, Ambika, et al.
(författare)
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Insights Into How Heme Reduction Potentials Modulate Enzymatic Activities of a Myoglobin-based Functional Oxidase
- 2017
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 56:23, s. 6622-6626
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Björck, Markus L., et al.
(författare)
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Reaction of S-cerevisiae mitochondria with ligands : Kinetics of CO and O-2 binding to flavohemoglobin and cytochrome c oxidase
- 2017
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1858:2, s. 182-188
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Blomberg, Margareta R. A., et al.
(författare)
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Mechanisms for enzymatic reduction of nitric oxide to nitrous oxide - A comparison between nitric oxide reductase and cytochrome c oxidase
- 2018
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1859:11, s. 1223-1234
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 11. |
- Blomberg, Margareta R. A., 1946-, et al.
(författare)
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Reduction of molecular oxygen in flavodiiron proteins - Catalytic mechanism and comparison to heme-copper oxidases
- 2024
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Ingår i: Journal of Inorganic Biochemistry. - 0162-0134 .- 1873-3344. ; 255
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Blomberg, Margareta R. A., 1946-, et al.
(författare)
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Reduction of Nitric Oxide to Nitrous Oxide in Flavodiiron Proteins : Catalytic Mechanism and Plausible Intermediates
- 2023
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 13:3, s. 2025-2038
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 13. |
- Blomberg, Margareta R. A., et al.
(författare)
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The mechanism for oxygen reduction in cytochrome c dependent nitric oxide reductase (cNOR) as obtained from a combination of theoretical and experimental results
- 2017
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1858:11, s. 884-894
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 14. |
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| 15. |
- Brzezinski, Peter, et al.
(författare)
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Intermediates generated during the reaction of reduced Rhodobacter sphaeroides cytochrome c oxidase with dioxygen
- 2013
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1827:7, s. 843-847
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 16. |
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| 17. |
- Brzezinski, Peter, et al.
(författare)
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Structure and Mechanism of Respiratory III-IV Supercomplexes in Bioenergetic Membranes
- 2021
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Ingår i: Chemical Reviews. - : American Chemical Society (ACS). - 0009-2665 .- 1520-6890. ; 121:15, s. 9644-9673
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 18. |
- Di Trani, Justin M., et al.
(författare)
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Structure of the bc1–cbb3 respiratory supercomplex from Pseudomonas aeruginosa
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 120:40
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Tidskriftsartikel (refereegranskat)abstract
- 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 bc1–cbb3 SCs also exist in other bacterial pathogens.
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| 19. |
- Eriksson, Jonas, 1986-
(författare)
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Kinetic assays for RNA-cleaving deoxyribozymes and other nucleases
- 2016
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis two different assays for real-time RNA-cleaving deoxyribozyme and general nuclease kinetics are presented. Previous publications on nuclease kinetic assays have been riddled with drawbacks of labeling, discontinuity, cost etc. To tackle some of the drawbacks two assays were developed; the first specifically for RNA-cleaving deoxyribozymes to allow real-time kinetic measurements independently of whether the deoxyribozyme has low or high levels of secondary structure and when cleaving a full length messenger RNA (mRNA) substrate; the second assay was developed as a means to measure kinetics of virtually any nuclease by utilizing the single ubiquitous phenomenon in nuclease cleavage, the exposure of a phosphate upon hydrolysis of the phosphate backbone.In Paper I the assay for RNA-cleaving deoxyribozyme kinetics is presented as a development of a previously published assay. The search for a fluorescent intercalating dye with more preferential properties than ethidium bromide resulted in PicoGreen. This dye allowed the assay to be used for deoxyribozymes with low and high levels of secondary structure as well as using full length mRNA substrates.Paper II presents the second assay of this thesis, an assay where phosphates exposed by nuclease cleavage are released from their products by phosphatases; the released inorganic phosphates are quantified in real-time by a biosensor. The assay allows for real-time kinetics without the use of labels (i.e. natural enzymes and substrates). Regardless of whether the nuclease was a protein, nucleic acid-based, an exo- or endonuclease, processive or single-target nuclease the assay suited them equally well.
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| 20. |
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| 21. |
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| 22. |
- Faxén, Kristina, 1957-
(författare)
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Active Transport of Ions across Biomembranes : A Kinetic Study of Cytochrome c Oxidase Reconstituted into Phospholipid Vesicles
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ion transport across membranes is of uttermost importance for us. It is the foundation for signaling of various kinds e.g. in the nerve-system. Furthermore, energy from photosynthesis and metabolism is conserved in electrochemical gradients across membranes, maintained by ion pumps. In this thesis I discuss mechanisms of how protons and other ions are translocated across biomembranes against their concentration gradients. I have studied one specific proton pump, cytochrome c oxidase (CytcO) and in the summary I also compare CytcO with two other pumps for which a wealth of structural and functional information has recently been obtained. The data in the articles presented in this thesis support a model were proton pumping can be achieved without simultaneous oxidation of heme a or electron transfer (paper I); where a proton is transferred to the catalytic site before the pump site is protonated (paper IV); and where proton release is preceded by a conformational change (paper II). These observations could be explained by a model involving a conformational change of the pump element, recently proposed from our laboratory1. Furthermore the results from the papers in this thesis show that proton uptake precedes proton release in D2O (paper II). The kinetics of electron transfers linked to proton pumping is solely determined by the pH on the N-side of the membrane (paper III). Finally Zn2+ added on the P-side of the membrane inhibits a specific reaction step (paper IV). In the three pumps described here conformational changes, modulating ion affinities, and the opening and closing of gates, seem to be involved in driving the ions across the membrane. 1. Brzezinski, P. & Larsson, G. (2003) Biochim. Biophys. Acta 1605, 1-13.
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| 23. |
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| 24. |
- Fedotovskaya, Olga, et al.
(författare)
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Identification of a cytochrome bc1-aa3 supercomplex in Rhodobacter sphaeroides
- 2021
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1862:8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 25. |
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| 26. |
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| 27. |
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| 28. |
- Flock, Ulrika, et al.
(författare)
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Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase
- 2009
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Ingår i: Journal of Inorganic Biochemistry. - : Elsevier BV. - 0162-0134 .- 1873-3344. ; 103:5, s. 845-850
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 29. |
- Flock, Ulrika, 1970-
(författare)
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Nitric Oxide Reductase from Paracoccus denitrificans : A Proton Transfer Pathway from the “Wrong” Side
- 2008
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Denitrification is an anaerobic process performed by several soil bacteria as an alternative to aerobic respiration. A key-step in denitrification (the N-N-bond is made) is catalyzed by nitric oxide reductase (NOR); 2NO + 2e- + 2H+ → N2O + H2O. NOR from Paracoccus denitrificans is a member of the heme copper oxidase superfamily (HCuOs), where the mitochondrial cytochrome c oxidase is the classical example. NOR is situated in the cytoplasmic membrane and can, as a side reaction, catalyze the reduction of oxygen to water.NORs have properties that make them divergent members of the HCuOs; the reactions they catalyze are not electrogenic and they do not pump protons. They also have five strictly conserved glutamates in their catalytic subunit (NorB) that are not conserved in the ‘classical’ HCuOs. It has been asked whether the protons used in the reaction really come from the periplasm and if so how do the protons proceed through the protein into the catalytic site?In order to find out whether the protons are taken from the periplasm or the cytoplasm and in order to pinpoint the proton-route in NorB, we studied electron- and proton transfer during a single- as well as multiple turnovers, using time resolved optical spectroscopy. Wild type NOR and several variants of the five conserved glutamates were investigated in their solubilised form or/and reconstituted into vesicles.The results demonstrate that protons needed for the reaction indeed are taken from the periplasm and that all but one of the conserved glutamates are crucial for the oxidative phase of the reaction that is limited by proton uptake to the active site.In this thesis it is proposed, using a model of NorB, that two of the glutamates are located at the entrance of the proton pathway which also contains two of the other glutamates close to the active site.
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| 30. |
- Gonska, Nathalie, 1985-, et al.
(författare)
-
Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 31. |
- Gonska, Nathalie, et al.
(författare)
-
Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme
- 2018
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 32. |
- Gonska, Nathalie, 1985-
(författare)
-
Proton pathways in energy conversion : K-pathway analogs in O2- and NO-reductases
- 2017
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic.Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction.The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved.
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| 33. |
- Graf, Simone, et al.
(författare)
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Rapid Electron Transfer within the III-IV Supercomplex in Corynebacterium glutamicum
- 2016
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 34. |
- Huang, Yafei, et al.
(författare)
-
Substrate binding and the catalytic reactions in cbb3-type oxidases : the lipid membrane modulates ligand binding
- 2010
-
Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434 .- 0005-2728. ; 1797:6-7, s. 724-31
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 35. |
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| 36. |
- Huang, Yafei, et al.
(författare)
-
Vectorial proton transfer coupled to reduction of O2 and NO by a heme-copper oxidase
- 2008
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:51, s. 20257-20262
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 37. |
- Kahle, Maximilian, 1986-
(författare)
-
Assembly and Function of Nitric Oxide Reductase from Paracoccus denitrificans
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study.Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR.Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this protein to investigate the role of FeB in the mechanism of nitric oxide and oxygen reduction of cNOR and compared our results to computational studies of the enzyme published recently.In vitro studies of membrane proteins, such as cNOR, are challenging because their function often depends on the interaction with a biological membrane and specific phospholipids. We used two different membrane mimetic systems, lipid nanodiscs and proteoliposomes, to study the effect of a membrane environment on the function of detergent-solubilized cNOR. Our results indicate that the membrane bilayer of lipid nanodiscs and proteoliposomes, even when assembled using the same lipids, has different properties with measurable effects on cNOR function.
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| 38. |
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| 39. |
|
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| 40. |
- Kahle, Maximilian, et al.
(författare)
-
Insights into the mechanism of nitric oxide reductase from a Fe-B-depleted variant
- 2019
-
Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 593:12, s. 1351-1359
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 41. |
- Kahle, Maximilian, 1986-, et al.
(författare)
-
Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins
- 2023
-
Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 21
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 42. |
- Kahle, Maximilian, et al.
(författare)
-
The insertion of the non-heme Fe-B cofactor into nitric oxide reductase from P. denitrificans depends on NorQ and NorD accessory proteins
- 2018
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1859:10, s. 1051-1058
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 43. |
- Knopp, Michael, et al.
(författare)
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De Novo Emergence of Peptides That Confer Antibiotic Resistance
- 2019
-
Ingår i: mBio. - : AMER SOC MICROBIOLOGY. - 2161-2129 .- 2150-7511. ; 10:3
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 44. |
- Król, Sylwia, et al.
(författare)
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Electron and proton transfer in the M. smegmatis III2IV2 supercomplex
- 2022
-
Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1863:7
-
Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 45. |
- Lachmann, Peter, 1980-
(författare)
-
Electron and Proton Transfer in Nitric Oxide Reductase : NO Binding, NO Reduction and no Pumping
- 2009
-
Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two electronreduction of NO to N2O (2NO + 2H+ + 2e- → N2O + H2O) as part of the process ofdenitrification, the step-wise reduction of nitrate to dinitogen. The NOR-catalyzedreaction is central in the nitrogen cycle, since in this step the N=N double bond isformed. NOR is a deviant heme copper oxidase, located in the cytoplasmic membrane,containing four redox active cofactors. Like cytochrome c oxidase (CcO), NOR canreduce oxygen to water as a side reaction, but in contrast to CcO it does not contributeto the proton motive force that drives the conversion of ADP to ATP by ATP synthase.The active site in the catalytic subunit NorB consists of a non-heme iron FeB and a hemeb3 that are anti-ferromagnetically coupled. Additionally a low-spin heme b in NorB isinvolved in accepting electrons from heme c of NorC, a membrane anchored cytochromec, which is the second subunit of the purified NorBC heterodimer.We have studied the terminal region of the proton entry channel and possible ligands tothe binuclear active site of NOR using the flow-flash technique and could demonstratethat the putative proton channel residues Glu(E)198 and E267 in NorB are essential forproton uptake. We propose that they define the terminal proton channel region close tothe binuclear site. An alanine variant of the fully conserved amino acid residue E202 ofNOR that, according to the model of NOR (47), is located in the vicinity to the active site,is neither essential for catalytic activity nor integrity of the active site.Furthermore, we were able to demonstrate the [NO] dependency of NOR in the reactionbetween fully reduced protein and NO using the flow-flash technique (21, 24). Thebinding of NO to the fully reduced enzyme is clearly concentration dependent,inconsistent with a previously proposed obligatory binding of NO first to FeB before itligates to heme b3, where it, in the first turnover, is reduced by the electrons from theactive site. Further oxidation involves electron transfer from the low-spin hemes, which isaccelerated at lower [NO]. This acceleration at lower substrate concentration is evenlarger at decreased pH. We could demonstrate that substrate inhibition, observed insteady-state measurements, occurs already on oxidizing the fully reduced enzyme,indicating that NO binds to its inhibitory site before electrons can redistribute to theactive site from the low-spin hemes.
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| 46. |
- Lachmann, Peter, 1980-
(författare)
-
Kinetics of proton and electron transfer in heme-copper oxidases
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heme-copper oxidases are transmembrane proteins that are found in aerobic and anaerobic respiratory chains. During aerobic respiration, these enzymes reduce dioxygen to water. The energy released in the reaction is used to transport protons across a biological membrane. Stored as proton electrochemical gradient, the energy can be used to regenerate ATP. It is known that aa3 oxidases, which are the most common oxidases, transport pumped protons and protons used for the catalytic reaction using two proton pathways. However, the molecular mechanism of pumping is still being debated.When oxygen is available in very small quantities, oxygen reductases with high affinity for oxygen are expressed by organisms like Thermus thermophilus. The proton pumping mechanism in the ba3 oxidase is slightly different from that of aa3 oxidases as this enzyme only uses a single proton uptake pathway. Here we analyzed the reaction mechanism of ba3 oxidase and found evidence that the first proton taken up by the four-electron reduced ba3 oxidase is transferred to a site distant from the catalytic site, the pump site, and that only every second proton taken up from solution is pumped. Data obtained from studies using site-directed mutagenesis and flow-flash spectroscopy suggest a probable location of the pump site.Under anaerobic conditions, some organisms are able to generate a proton- motive force using nitrate and nitrite as electron acceptors. In this process, the cytotoxic reaction intermediate nitric oxide is produced. Nitric oxide reductase (NOR), a deviant heme-copper oxidase that reduces NO to the rather harmless N2O, does not pump any protons. The catalytic mechanism of nitric oxide reduction by NOR is very poorly understood.Here we demonstrate that substrate inhibition, which occurs in NOR from Paracoccus denitrificans above 5 μM NO, can already be observed before the electrons from the low-spin hemes re-distribute to the active site. Furthermore, we found that a single specific proton pathway is used for proton-transfer leading from the periplasm to the active site.
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| 47. |
- Lachmann, Peter, et al.
(författare)
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Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase
- 2010
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:33, s. 25531-25537
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 48. |
- Lee, Hyun Ju, et al.
(författare)
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Entrance of the proton pathway in cbb3-type heme-copper oxidases
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:43, s. 17661-6
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 49. |
- Lee, Hyun Ju, et al.
(författare)
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Functional proton transfer pathways in the heme-copper oxidase superfamily
- 2012
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1817:4, s. 537-544
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Forskningsöversikt (refereegranskat)abstract
- Heme-copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-. B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases.
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| 50. |
- Lee, Hyun Ju, et al.
(författare)
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The heme-copper oxidase superfamily shares a Zn2+-binding motif at the entrance to a proton pathway
- 2013
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 587:6, s. 770-774
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Tidskriftsartikel (refereegranskat)abstract
- Heme-copper oxidases (HCuOs) catalyse the reduction of oxygen, using the liberated free energy to maintain a proton-motive force across the membrane. In the mitochondrial-like A-type HCuOs, binding of heavy metal ions at the surface of the protein inhibits proton transfer. In bacterial C-type oxidases, the entry point to the proton pathway is on an accessory subunit unrelated to any subunit in A-type HCuOs. Despite this, we show here that heavy metal ions such as Zn2+ inhibit O-2-reduction very similarly in C-type as in A-type HCuOs, and furthermore that the binding site shares the same Glu-His motif. (C) 2013 Federation of European Biochemical Societies.
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