| 1. |
- Lachmann, Peter, 1980-
(författare)
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Electron and Proton Transfer in Nitric Oxide Reductase : NO Binding, NO Reduction and no Pumping
- 2009
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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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| 2. |
- Persson Waye, Kerstin, 1959, et al.
(författare)
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Adopting a child perspective for exposome research on mental health and cognitive development - Conceptualisation and opportunities.
- 2023
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Ingår i: Environmental research. - 0013-9351 .- 1096-0953. ; 239:Pt 1
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Tidskriftsartikel (refereegranskat)abstract
- Mental disorders among children and adolescents pose a significant global challenge. The exposome framework covering the totality of internal, social and physical exposures over a lifetime provides opportunities to better understand the causes of and processes related to mental health, and cognitive functioning. The paper presents a conceptual framework on exposome, mental health, and cognitive development in children and adolescents, with potential mediating pathways, providing a possibility for interventions along the life course. The paper underscores the significance of adopting a child perspective to the exposome, acknowledging children's specific vulnerability, including differential exposures, susceptibility of effects and capacity to respond; their susceptibility during development and growth, highlighting neurodevelopmental processes from conception to young adulthood that are highly sensitive to external exposures. Further, critical periods when exposures may have significant effects on a child's development and future health are addressed. The paper stresses that children's behaviour, physiology, activity pattern and place for activities make them differently vulnerable to environmental pollutants, and calls for child-specific assessment methods, currently lacking within today's health frameworks. The importance of understanding the interplay between structure and agency is emphasized, where agency is guided by social structures and practices and vice-versa. An intersectional approach that acknowledges the interplay of social and physical exposures as well as a global and rural perspective on exposome is further pointed out. To advance the exposome field, interdisciplinary efforts that involve multiple scientific disciplines are crucial. By adopting a child perspective and incorporating an exposome approach, we can gain a comprehensive understanding of how exposures impact children's mental health and cognitive development leading to better outcomes.
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| 3. |
- Prasad, Rashmi B., et al.
(författare)
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Excess maternal transmission of variants in the THADA gene to offspring with type 2 diabetes
- 2016
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Ingår i: Diabetologia. - : Springer Science and Business Media LLC. - 0012-186X .- 1432-0428. ; 59:8, s. 1702-1713
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Tidskriftsartikel (refereegranskat)abstract
- Aims/hypothesis: Genome-wide association studies (GWAS) have identified more than 65 genetic loci associated with risk of type 2 diabetes. However, the contribution of distorted parental transmission of alleles to risk of type 2 diabetes has been mostly unexplored. Our goal was therefore to search for parent-of-origin effects (POE) among type 2 diabetes loci in families. Methods: Families from the Botnia study (n = 4,211, 1,083 families) were genotyped for 72 single-nucleotide polymorphisms (SNPs) associated with type 2 diabetes and assessed for POE on type 2 diabetes. The family-based Hungarian Transdanubian Biobank (HTB) (n = 1,463, >135 families) was used to replicate SNPs showing POE. Association of type 2 diabetes loci within families was also tested. Results: Three loci showed nominal POE, including the previously reported variants in KCNQ1, for type 2 diabetes in families from Botnia (rs2237895: pPOE = 0.037), which can be considered positive controls. The strongest POE was seen for rs7578597 SNP in the THADA gene, showing excess transmission of the maternal risk allele T to diabetic offspring (Botnia: pPOE = 0.01; HTB pPOE = 0.045). These data are consistent with previous evidence of allelic imbalance for expression in islets, suggesting that the THADA gene can be imprinted in a POE-specific fashion. Five CpG sites, including those flanking rs7578597, showed differential methylation between diabetic and non-diabetic donor islets. Conclusions/interpretation: Taken together, the data emphasise the need for genetic studies to consider from which parent an offspring has inherited a susceptibility allele.
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| 4. |
- von Ballmoos, Christoph, et al.
(författare)
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Mutation of a single residue in the ba(3) oxidase specifically impairs protonation of the pump site
- 2015
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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. ; 112:11, s. 3397-3402
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Tidskriftsartikel (refereegranskat)abstract
- The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O-2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba(3) oxidase where a putative pump site was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O-2 reduction. The results from our studies show that proton uptake to the pump site (time constant similar to 65 mu s in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of similar to 1.2 ms was slowed to similar to 8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba(3) cytochrome c oxidase.
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| 5. |
- von Ballmoos, Christoph, et al.
(författare)
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Timing of Electron and Proton Transfer in the ba(3) Cytochrome c Oxidase from Thermus thermophilus
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:22, s. 4507-4517
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Tidskriftsartikel (refereegranskat)abstract
- Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O-2 to H2O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O-2 reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H+/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H+/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba3 oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a proton-loading site (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.
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| 6. |
- 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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| 7. |
- Lachmann, Peter, 1980-
(författare)
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Kinetics of proton and electron transfer in heme-copper oxidases
- 2015
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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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| 8. |
- Lachmann, Peter, et al.
(författare)
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Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase
- 2010
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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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| 9. |
- Máñez Costa, Maria, et al.
(författare)
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Co-production of Climate Services : A diversity of approaches and good practice from the ERA4CS projects (2017–2021)
- 2022
-
Rapport (övrigt vetenskapligt/konstnärligt)abstract
- This guide presents a joint effort of projects funded under the European Research Area for Climate Services (ERA4CS) (http://www.jpi-climate.eu/ERA4CS), a co- funded action initiated by JPI Climate with co-funding by the European Union (Grant 690462), 15 national public Research Funding Organisations (RFOs), and 30 Research Performing Organisations (RPOs) from 18 European countries. This guide sets out to increase the understanding of different pathways, methods, and approaches to improve knowledge co-production of climate services with users as a value-added activity of the ERA4CS Programme.Reflecting on the experiences of 16 of the 26 projects funded under ERA4CS, this guide aims to define and recommend good practices for transdisciplinary knowledge co-production of climate services to researchers, users, funding agencies, and private sector service providers. Drawing on responses from ERA4CS project teams to a questionnaire and interviews, this guide maps the diversity of methods for stakeholder identification, involvement, and engagement. It also conducts an analysis of methods, tools, and mechanisms for engagement as well as evaluation of co-production processes.This guide presents and discusses good practice examples based on the review of the ERA4CS projects, identifying enablers and barriers for key elements in climate service co-production processes. These were: namely (i) Forms of Engagement; (ii) Entry Points for Engagement; and, (iii) Intensity of Involvement. It further outlines key ingredients to enhance the quality of co-producing climate services with users and stakeholders.Based on the analysis of the lessons learned from ERA4CS projects, as well as a review of key concepts in the recent literature on climate service co-production, we provide a set of recommendations for researchers, users, funders and private sector providers of climate services.
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| 10. |
- Rinaldo-Matthis, Agnes, et al.
(författare)
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Pre-Steady-State Kinetic Characterization of Thiolate Anion Formation in Human Leukotriene C-4 Synthase
- 2012
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 51:4, s. 848-856
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Tidskriftsartikel (refereegranskat)abstract
- Human leukotriene C-4 synthase (hLTC4S) is an integral membrane protein that catalyzes the committed step in the biosynthesis of cysteinyl-leukotrienes, i.e., formation of leukotriene C-4 (LTC4). This molecule, together with its metabolites LTD4 and LTE4, induces inflammatory responses, particularly in asthma, and thus, the enzyme is an attractive drug target. During the catalytic cycle, glutathione (GSH) is activated by hLTC4S that forms a nucleophilic thiolate anion that will attack LTA(4), presumably according to an S(N)2 reaction to form LTC4. We observed that GSH thiolate anion formation is rapid and occurs at all three monomers of the homotrimer and is concomitant with stoichiometric release of protons to the medium. The pK(a) (5.9) for enzyme-bound GSH thiol and the rate of thiolate formation were determined (k(obs) = 200 s(-1)). Taking advantage of a strong competitive inhibitor, glutathionesulfonic acid, shown here by crystallography to bind in the same location as GSH, we determined the overall dissociation constant (K-d(GS) = 14.3 mu M). The release of the thiolate was assessed using a GSH release experiment (1.3 s(-1)). Taken together, these data establish that thiolate anion formation in hLTC4S is not the rate-limiting step for the overall reaction of LTC4 production (k(cat) = 26 s(-1)), and compared to the related microsomal glutathione transferase 1, which displays very slow GSH thiolate anion formation and one-third of the sites reactivity, hLTC4S has evolved a different catalytic mechanism.
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