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A room temperature stable Y(ii)bis(amide) has been prepared and fully characterized. The complex reacts with tert-butylisocyanide to make a rare example of a transition metal isocyanide, CN–Y(NHAr*)2.

Perspective from Difluoromethylornithine Plus Sulindac for the Prevention of Sporadic Colorectal Adenomas: A Randomized Placebo-Controlled, Double-Blind Trial

While reform of Canada's electoral system has not yet occurred, it has been an ever-present, ever-potent topic in Canadian political science since the middle of the 20th Century. While there are aspects of First-Past-the-Post (FPTP) to be admired, its fundamental flaws cannot be ignored; the system encourages parties to exacerbate sectionalism, leaves far too many voters unrepresented, and too often allows for uncooperative governments. Mixed-Member Proportional (MMP), a form of Proportional Representation (PR), possesses the best aspect of FPTP, local representation and accountability, and amends its most serious flaws. MMP represents all votes cast, attenuates sectionalism, and creates diverse legislatures that incentivize cross-party cooperation far more than FPTP does currently. Canada is nearly a perfect country for MMP, and this system should be implemented for our federal elections.

The title article serves as a lasting product of the 25-year scientific collaboration between Jack Peisach, a biophysical chemist at the Albert Einstein College of Medicine, and William E. Blumberg, a physicist at Bell Telephone Laboratories.

Introducing spin onto organic ligands that are coordinated to rare earth metal ions allows direct exchange with metal spin centres. This is particularly relevant for the deeply buried 4f-orbitals of the lanthanide ions that can give rise to unparalleled magnetic properties. For efficacy of exchange coupling, the donor atoms of the radical ligand require high-spin density. Such molecules are extremely rare owing to their reactive nature that renders isolation and purification difficult. Here, we demonstrate that a 2,2′-azopyridyl (abpy) radical (S = 1/2) bound to the rare earth metal yttrium can be realized. This molecule represents the first rare earth metal complex containing an abpy radical and is unambigously characterized by X-ray crystallography, NMR, UV-Vis-NIR, and IR spectroscopy. In addition, the most stable isotope 89Y with a natural abundance of 100% and a nuclear spin of ½ allows an in-depth analysis of the yttrium–radical complex via EPR and HYSCORE spectroscopy. Further insight into the electronic ground state of the radical azobispyridine-coordinated metal complex was realized through unrestricted DFT calculations, which suggests that the unpaired spin density of the SOMO is heavily localized on the azo and pyridyl nitrogen atoms. The experimental results are supported by NBO calculations and give a comprehensive picture of the spin density of the azopyridyl ancillary ligand. This unexplored azopyridyl radical anion in heavy element chemistry bears crucial implications for the design of molecule-based magnets particularly comprising anisotropic lanthanide ions., Unambiguous characterization of the first 2,2′-azobispyridine radical-containing rare earth metal complex through X-ray crystallography, DFT computations, EPR and HYSCORE spectroscopy.

Background Household air pollution adversely affects human health and the environment, yet more than 40% of the world still depends on solid cooking fuels. The House Air Pollution Intervention Network (HAPIN) randomized controlled trial is assessing the health effects of a liquefied petroleum gas (LPG) stove and 18-month supply of free fuel in 3,200 households in rural Guatemala, India, Peru, and Rwanda. Aims We conducted formative research in Guatemala to create visual messages that support the sustained, exclusive use of LPG in HAPIN intervention households. Method We conducted ethnographic research, including direct observation ( n = 36), in-depth ( n = 18), and semistructured ( n = 6) interviews, and 24 focus group discussions ( n = 96) to understand participants’ experience with LPG. Sixty participants were selected from a pilot study of LPG stove and 2-months of free fuel to assess the acceptability and use of LPG. Emergent themes were used to create visual messages based on observations and interviews in 40 households; messages were tested and revised in focus group discussions with 20 households. Results We identified 50 codes related to household air pollution and stoves; these were reduced into 24 themes relevant to LPG stoves, prioritizing 12 for calendars. Messages addressed fear and reluctance to use LPG; preference of wood stoves for cooking traditional foods; sustainability and accessibility of fuel; association between health outcomes and household air pollution; and the need for inspirational and aspirational messages. Discussion We created a flip chart and calendar illustrating themes to promote exclusive LPG use in HAPIN intervention households.

In the early 20th Century Canada saw the rise of a prominent socialist movement led by the Co-operative Commonwealth Federation (CCF). The CCF's influence on Canadian politics was essential to the creation of Canada's modern political ideology, which can be described as reform liberal. This ideology took hold due to the pressure that the CCF exerted on the two major federal parties, which could both be characterized as classical liberal. Due to the settlement pattern of the prairies and the actions of the federal government in response to the Great Depression, the CCF was able to secure a strong support base that propelled it to federal politics and allowed it to form a provincial government in Saskatchewan. Though it never formed a federal government, the CCF pushed for old age pension, reforms of corporate taxation, and Medicare. As a provincial actor and a "third force" upon the two ruling federal parties, the CCF and its successor the New Democratic Party’s contributions to Canadian identity and policy are beyond dispute.

Sulfur-insertion reactions are essential for the biosynthesis of several cellular metabolites, including enzyme cofactors. In Lactobacillus plantarum, a sulfur-containing nickel-pincer nucleotide (NPN) cofactor is used as a coenzyme of lactic acid racemase, LarA. During NPN biosynthesis in L. plantarum, sulfur is transferred to a nicotinic acid-derived substrate by LarE, which sacrifices the sulfur atom of its single cysteinyl side chain, forming a dehydroalanine residue. Most LarE homologs contain three conserved cysteine residues that are predicted to cluster at the active site; however, the function of this cysteine cluster is unclear. In this study, we characterized LarE from Thermotoga maritima (LarE

Proton Hyperfine Sublevel Correlation ((1)H-HYSCORE) experiments have been used to probe the ligation structure of the Fe(II) active site of taurine:2-oxoglutarate dioxygenase (TauD), a non-heme Fe(II) hydroxylase. To facilitate Electron Paramagnetic Resonance (EPR) experiments, Fe(II) derivatives of the enzyme were studied using nitric oxide as a substitute for molecular oxygen. The addition of NO to the enzyme yields an S = 3/2 {FeNO}(7) paramagnetic center characterized by nearly axial EPR spectra with g(⊥) = 4 and g(||) = 2. Using results from (i) an X-ray crystallographic study of TauD crystallized under anaerobic conditions in the presence of both cosubstrate 2-oxoglutarate and substrate taurine, (ii) a published theoretical description of the {FeNO}(7) derivative of this form of the enzyme, and (iii) previous (2)H-Electron Spin Echo Envelope Modulation (ESEEM) studies, we were able to assign the proton cross peaks detected in orientation-selected (1)H-HYSCORE spectra. Discrete contributions from the protons of two coordinated histidine ligands were resolved. If substrate taurine is absent from the complex, orientation-selective HYSCORE spectra show cross peaks that are less resolved and when combined with information obtained from continuous wave EPR, support an alternate binding scheme for 2-oxoglutarate. HYSCORE studies of TauD in the absence of 2-oxoglutarate show additional (1)H cross peaks that can be assigned to two distinct bound water molecules. In addition, (1)H and (14)N cross peaks that arise from the coordinated histidine side chains show a change in NO coordination for this species. For all of the TauD species, (1)H hyperfine couplings and their orientations are sensitive to the detailed electronic structure of the {FeNO}(7) center.

Summary Pneumonia is a leading killer of children younger than 5 years despite high vaccination coverage, improved nutrition, and widespread implementation of the Integrated Management of Childhood Illnesses algorithm. Assessing the effect of interventions on childhood pneumonia is challenging because the choice of case definition and surveillance approach can affect the identification of pneumonia substantially. In anticipation of an intervention trial aimed to reduce childhood pneumonia by lowering household air pollution, we created a working group to provide recommendations regarding study design and implementation. We suggest to, first, select a standard case definition that combines acute (≤14 days) respiratory symptoms and signs and general danger signs with ancillary tests (such as chest imaging and pulse oximetry) to improve pneumonia identification; second, to prioritise active hospital-based pneumonia surveillance over passive case finding or home-based surveillance to reduce the risk of non-differential misclassification of pneumonia and, as a result, a reduced effect size in a randomised trial; and, lastly, to consider longitudinal follow-up of children younger than 1 year, as this age group has the highest incidence of severe pneumonia.

The recent demand of multifunctional materials and devices for advanced applications in energy conversion and data storage resulted into a revival of multiferroics, that is, materials characterized...

Lactate racemase (LarA) of Lactobacillus plantarum contains a novel organometallic cofactor with nickel coordinated to a covalently tethered pincer ligand, pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide, but its function in the enzyme mechanism has not been elucidated. This study presents direct evidence that the nickel-pincer cofactor facilitates a proton-coupled hydride transfer (PCHT) mechanism during LarA-catalyzed lactate racemization. No signal was detected by electron paramagnetic resonance spectroscopy for LarA in the absence or presence of substrate, consistent with a +2 metal oxidation state and inconsistent with a previously proposed proton-coupled electron transfer mechanism. Pyruvate, the predicted intermediate for a PCHT mechanism, was observed in quenched solutions of LarA. A normal substrate kinetic isotope effect (kH/kD of 3.11 ± 0.17) was established using 2-α-2H-lactate, further supporting a PCHT mechanism. UV–visible spectroscopy revealed a lactate-induced perturbation of ...

Reduction of IU(NHAriPr6)2 (AriPr6 = 2,6-(2,4,6-iPr3C6H2)2C6H3) results in a rare example of a U(II) complex, U(NHAriPr6)2, and the first example that is a neutral species. Here, we show spectroscopic and magnetic studies that suggest a 5f46d0 valence electronic configuration for uranium, along with characterization of related U(III) complexes.

Epoxy coatings are used in a wide variety of applications due to their excellent chemical, thermal, and mechanical properties. However, their susceptibility to UV degradation has limited their use in exterior applications. Usually, organic UV absorbers and stabilizers are added to epoxy systems to improve their UV stability, but their performance decreases over time due to the degradation and loss of organic UV-stabilizers. In this study, a novel method was developed to encapsulate organic UV stabilizers and lignin (as a natural UV absorber) into halloysite nanotubes (HNTs). To ensure successful encapsulation, the pristine and filled halloysite nanotubes were characterized quantitatively, using a thermogravimetric analyzer (TGA), and qualitatively with X-ray photoelectron spectroscopy (XPS). Then, encapsulated nanotubes (HNTs) were added to an epoxy system (1−3 wt%) and their efficacy was evaluated before and after 840 h of accelerated weathering. Changes in physical, chemical, and thermal properties of coatings were measured using a spectrophotometer, field emission scanning electron microscopy (FE-SEM), electron paramagnetic resonance spectroscopy (EPR), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). The results showed that epoxy samples containing 2 wt% HNT-encapsulated with organic UV-stabilizers, and samples containing 1 wt% HNTs encapsulated with lignin had significantly higher UV stability than epoxy resin prepared with the same concentration of these individual components (UV-stabilizers or lignin). This study demonstrates the efficacy of encapsulated HNTs, with UV stabilizers or lignin, in improving UV stability and extending service life of epoxy coatings. Nanoclay (HNT) loaded with lignin, a natural polymer, can be used as an effective UV-absorber to increase the weathering performance of epoxy resins.

A wide range of spectroscopic approaches have been used to interrogate the mononuclear iron metallocenter in 2-oxoglutarate (2OG)-dependent oxygenases. The results from these spectroscopic studies have provided valuable insights into the structural changes at the active site during substrate binding and catalysis, thus providing critical information that complements investigations of these enzymes by X-ray crystallography, biochemical, and computational approaches. This mini-review highlights taurine hydroxylase (taurine:2OG dioxygenase, TauD) as a case study to illustrate the wealth of knowledge that can be generated by applying a diverse array of spectroscopic investigations to a single enzyme. In particular, electronic absorption, circular dichroism, magnetic circular dichroism, conventional and pulse electron paramagnetic, Mössbauer, X-ray absorption, and resonance Raman methods have been exploited to uncover the properties of the metal site in TauD.

The remarkable rise of Scottish nationalism in the last few decades has been paralleled by the publication of a host of books and articles focusing on Scotland’s role in the rise and decline of the...

The journal The Lancet recently published a countdown on health and climate change. Attention was focused solely on humans. However, animals, including wildlife, livestock and pets, may also be impacted by climate change. Complementary to the high relevance of awareness rising for protecting humans against climate change, here we present a One Health approach, which aims at the simultaneous protection of humans, animals and the environment from climate change impacts (climate change adaptation). We postulate that integrated approaches save human and animal lives and reduce costs when compared to public and animal health sectors working separately. A One Health approach to climate change adaptation may significantly contribute to food security with emphasis on animal source foods, extensive livestock systems, particularly ruminant livestock, environmental sanitation, and steps towards regional and global integrated syndromic surveillance and response systems. The cost of outbreaks of emerging vector-borne zoonotic pathogens may be much lower if they are detected early in the vector or in livestock rather than later in humans. Therefore, integrated community-based surveillance of zoonoses is a promising avenue to reduce health effects of climate change., FEMS Microbiology Letters, 365 (11), ISSN:0378-1097, ISSN:1574-6968, ISSN:0168-6496, ISSN:0920-8534, ISSN:0168-6445

Terence Ranger was one of the most stimulating and influential African historians of the last half century. A scholar of remarkable energy, Terry, as he was universally known, had the reputation of...

Electron paramagnetic resonance (EPR) experiments were done on a series of S = (3)/2 ferrous nitrosyl model complexes prepared with chelating ligands that mimic the 2-His-1-carboxylate facial triad iron binding motif of the mononuclear nonheme iron oxidases. These complexes formed a comparative family, {FeNO}(7)(N2Ox)(H2O)3-x with x = 1-3, where the labile coordination sites for the binding of NO and solvent water were fac for x = 1 and cis for x = 2. The continuous-wave EPR spectra of these three complexes were typical of high-spin S = (3)/2 transition-metal ions with resonances near g = 4 and 2. Orientation-selective hyperfine sublevel correlation (HYSCORE) spectra revealed cross peaks arising from the protons of coordinated water in a clean spectral window from g = 3.0 to 2.3. These cross peaks were absent for the {FeNO}(7)(N2O3) complex. HYSCORE spectra were analyzed using a straightforward model for defining the spin Hamiltonian parameters of bound water and showed that, for the {FeNO}(7)(N2O2)(H2O) complex, a single water conformer with an isotropic hyperfine coupling, Aiso = 0.0 ± 0.3 MHz, and a dipolar coupling of T = 4.8 ± 0.2 MHz could account for the data. For the {FeNO}(7)(N2O)(H2O)2 complex, the HYSCORE cross peaks assigned to coordinated water showed more frequency dispersion and were analyzed with discrete orientations and hyperfine couplings for the two water molecules that accounted for the observed orientation-selective contour shapes. The use of three-pulse electron spin echo envelope modulation (ESEEM) data to quantify the number of water ligands coordinated to the {FeNO}(7) centers was explored. For this aspect of the study, HYSCORE spectra were important for defining a spectral window where empirical integration of ESEEM spectra would be the most accurate.

Tyrosine hydroxylase is a mononuclear non-heme iron monooxygenase found in the central nervous system that catalyzes the hydroxylation of tyrosine to yield L-3,4-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. Catalysis requires the binding of tyrosine, a tetrahydropterin, and O₂ at an active site that consists of a ferrous ion coordinated facially by the side chains of two histidines and a glutamate. We used nitric oxide as a surrogate for O₂ to poise the active site iron in an S = ³/₂ {FeNO}⁷ form that is amenable to electron paramagnetic resonance (EPR) spectroscopy. The pulsed EPR method of hyperfine sublevel correlation (HYSCORE) spectroscopy was then used to probe the ligands at the remaining labile coordination sites on iron. For the complex formed by the addition of tyrosine and nitric oxide, TyrH/NO/Tyr, orientation-selective HYSCORE studies provided evidence of the coordination of one H₂O molecule characterized by proton isotropic hyperfine couplings (A(iso) = 0.0 ± 0.3 MHz) and dipolar couplings (T = 4.4 and 4.5 ± 0.2 MHz). These data show complex HYSCORE cross peak contours that required the addition of a third coupled proton, characterized by an A(iso) of 2.0 MHz and a T of 3.8 MHz, to the analysis. This proton hyperfine coupling differed from those measured previously for H₂O bound to {FeNO}⁷ model complexes and was assigned to a hydroxide ligand. For the complex formed by the addition of tyrosine, 6-methyltetrahydropterin, and NO, TyrH/NO/Tyr/6-MPH₄, the HYSCORE cross peaks attributed to H₂O and OH⁻ for the TyrH/NO/Tyr complex were replaced by a cross peak due to a single proton characterized by an A(iso) of 0.0 MHz and a dipolar coupling (T = 3.8 MHz). This interaction was assigned to the N₅ proton of the reduced pterin.

Numerous studies have reported the beneficial effects of antioxidants in human diseases. Among their biological effects, a majority of antioxidants scavenge reactive radicals in the body, thereby reducing oxidative stress that is associated with the pathogenesis of many diseases. Antioxidant dendrimers are a new class of potent antioxidant compounds reported recently. In this study, six polyphenol-based antioxidant dendrimers with or without electron donating groups (methoxy group) were synthesized in order to elucidate the influence of electron donating groups (EDG) on their antioxidant activities. Syringaldehyde (2 ortho methoxy groups), vanillin (1 ortho methoxy group), and 4-hydroxybenzaldehyde (0 methoxy group) were derivatized with propargylamine to form building blocks for the dendrimers. All the six dendrimers contain polyether cores, which were synthesized by attaching pentaerythritol and methyl α-D-glucopyranoside to in-house prepared spacer units. To prepare generation 1 antioxidant dendrimers, microwave energy and granulated metallic copper catalyst were used to link the cores and building blocks together via alkyne-azide 1,3-cycloaddition click chemistry. These reaction conditions resulted in high yields of the target dendrimers that were free from copper contamination. Based on DPPH antioxidant assay, antioxidant dendrimers decorated with syringaldehyde and vanillin exhibited over 70- and 170-fold increase in antioxidant activity compared to syringaldehyde and vanillin, respectively. The antioxidant activity of dendrimers increased with increasing number of EDG groups. Similar results were obtained when the dendrimers were used to protect DNA and human LDL against organic carbon and nitrogen-based free radicals. In addition, the antioxidant dendrimers did not show any pro-oxidant activity on DNA in the presence of physiological amounts of copper. Although the dendrimers showed potent antioxidant activities against carbon and nitrogen free radicals, EPR and DNA protection studies revealed lack of effectiveness of these dendrimers against hydroxyl radicals. The dendrimers were not cytotoxic to CHO-K1 cells.

Keywords: Common indicators; policy responses; prescription painkillers dependence; monitoring; reporting

We have investigated the stability of doxorubicin and daunorubicin complexes with Fe(3+) in aqueous solution. Doxorubicin and daunorubicin are anthracycline chemotherapeutic agents that differ structurally by the presence of a hydroxymethylketone functionality in doxorubicin versus a methyl ketone moiety in daunorubicin. We demonstrate that the presence of the hydroxyl group in doxorubicin enhances its 1:1 complexation with Fe(3+) relative to that seen for daunorubicin. We utilize UV-visible absorbance, circular dichroism, fluorescence and EPR spectroscopies, molecular dynamics, and semiempirical calculations to understand how the presence of an additional hydroxyl group affects the interactions of anthracyclines with Fe(3+). Our data indicate that the binding mode of iron in the complex is different for doxorubicin and daunorubicin.

This article introduces, reproduces, and contextualizes two letters written in 1938 from Edinburgh by Hastings Kamuzu Banda to Samuel C. Banda (Chief Mwase of Kasungu) and Ernest C. Matako. At the time Banda was studying in Edinburgh. The letters, from a period when sources on Banda are scarce, illuminate his political and educational thinking and his personal life and attitudes at the time. The article also discusses the archival context of the letters, which were found in the papers of Godfrey Wilson, the Director of the Rhodes-Livingstone Institute from 1938 to 1941. The copies were found in the course of research into the lives of the anthropologists Monica Wilson, nee Hunter, and Godfrey Wilson. Cet article introduit, reproduit, et contextualise deux lettres ecrites en 1938 a Edimbourg par Hastings Kamuzu Banda a Samuel C. Banda (Chef Mwase du Kasungu) et Ernest C. Matako. A l’epoque, Banda etudiait a Edimbourg. Les lettres, datant d’une periode ou les sources sur Banda sont rares, mettent en lumiere ses idees politiques et educatives, ainsi que ses attitudes et sa vie personnelle a ce moment-la. Cet article examine egalement le contexte d’archivage de ces lettres, qui furent trouvees dans les papiers de Godfrey Wilson, directeur de l’Institut Rhodes-Livingstone entre 1938 et 1941. Les copies furent trouvee au cours d’une recherche sur la vie des anthropologues Monica Wilson, nee Hunter, et de Godfrey Wilson.

This article explores the influence of Mau Mau and of the counter-insurgency strategies devised by the British in Kenya on the state of emergency declared in Nyasaland in March 1959. It focuses on the hitherto largely undisclosed history of Malawian detainees both in Gwelo and Marandellas prisons in Southern Rhodesia and in Kanjedza camp near Blantyre, where conditions were notably more severe. The Kenyan influence was particularly evident in the Pinney Plan of April 1959 which was predicated on the need for detainees to be ‘rehabilitated’ (cleansed of their mistaken beliefs) through a combination of hard work and confession. This assessment, however, was challenged in the Colonial Office partly as a result of the Hola Camp massacre in Kenya but also because of the publicity given to the mistreatment of detainees in Kanjedza. The extreme brutality of the early weeks was largely abandoned; at the same time detainees took control of much of the internal workings of Kanjedza. Much remains to be studied conce...

In this work, we investigated the oxidative modification of histidine residues induced by peroxidase and thiol oxidase activities of bovine copper-zinc superoxide dismutase (Cu-ZnSOD) using NMR and pulse EPR spectroscopy. 1D NMR and 2D-NOESY were used to determine the oxidative damage at the Zn(II) and Cu(II) active sites as well as at distant histidines. Results indicate that during treatment of SOD with hydrogen peroxide (H(2)O(2)) or cysteine in the absence of bicarbonate anion (HCO(3)(-)), both exchangeable and nonexchangeable protons were affected. Both His-44 and His-46 in the Cu(II) active site were oxidized based on the disappearance of NOESY cross-peaks between CH and NH resonances of the imidazole rings. In the Zn(II) site, only His-69, which is closer to His-44, was oxidatively modified. However, addition of HCO(3)(-) protected the active site His residues. Instead, resonances assigned to the His-41 residue, 11 Å away from the Cu(II) site, were completely abolished during both HCO(3)(-)-stimulated peroxidase activity and thiol oxidase activity in the presence of HCO(3)(-) . Additionally, ESEEM/HYSCORE and ENDOR studies of SOD treated with peroxide/Cys in the absence of HCO(3)(-) revealed that hyperfine couplings to the distal and directly coordinated nitrogens of the His-44 and His-46 ligands at the Cu(II) active site were modified. In the presence of HCO(3)(-), these modifications were absent. HCO(3)(-)-mediated, selective oxidative modification of histidines in SOD may be relevant to understanding the molecular mechanism of SOD peroxidase and thiol oxidase activities.

[Review] Richard Price, Making Empire: Colonial Encounters and the Creation of Imperial Rule in Nineteenth-Century South Africa (Cambridge University Press, Cambridge, 2008), xxix þ371 pp., £50 hardback, ISBN 978-0-521-88968-1; £18.99 paperback, ISBN 978-0-521-71819-6.

We examined the anion binding behavior of the Mg(Mn) site in cytochrome c oxidase to test a possible role of this center in proton pumping. Rhodobacter sphaeroides grown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. Due to its close proximity and a shared ligand, oxidized Cu(A) is spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine and R.s. oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when Cu(A) is oxidized. In the reduced structure, one water molecule in the vicinity of the Cu(A) ligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochrome c oxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochrome c oxidase. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.

Eukaryotic cytochrome c oxidase (CcO) and homologous prokaryotic forms of Rhodobacter and Paraccocus differ in the EPR spectrum of heme a. It was noted that a histidine ligand of heme a (H102) is hydrogen bonded to serine in Rhodobacter (S44) and Paraccocus CcOs, in contrast to glycine in the bovine enzyme. Mutation of S44 to glycine shifts the heme a EPR signal from g(z) = 2.82 to 2.86, closer to bovine heme a at 3.03, without modifying other properties. Mutation to aspartate, however, results in an oppositely shifted and split heme a EPR signal of g(z) = 2.72/2.78, accompanied by lower activity and drastically inhibited intrinsic electron transfer from CuA to heme a. This intrinsic rate is biphasic; the proportion that is slow is pH dependent, as is the relative intensity of the two EPR signal components. At pH 8, the heme a EPR signal at 2.72 is most intense, and the electron transfer rate (CuA to heme a) is 10-130 s(-1), compared to wild-type at 90,000 s(-1). At pH 5.5, the signal at 2.78 is intensified, and a biphasic rate is observed, 50% fast (approximately wild type) and 50% slow (90 s(-1)). The data support the prediction that the hydrogen-bonding partner of the histidine ligand of heme a is one determinant of the EPR spectral difference between bovine and bacterial CcO. We further demonstrate that the heme a redox potential can be dramatically altered by a nearby carboxyl, whose protonation leads to a proton-coupled electron transfer process.

Aromatic amino acid hydroxylases are members of a larger group of enzymes that use a mononuclear nonheme Fe center to catalyze a variety of thermodynamically challenging reactions in which O2 is used in the oxidative transformation of substrates. The hydroxylase enzymes are catalytically active in the ferrous oxidation state and are high-spin. To render the catalytic site EPR-active, we have used nitric oxide (NO) as a surrogate for substrate O2 to form an S=3/2 paramagnetic center. While the continuous-wave (cw)-EPR spectra of NO-enzyme adducts are rather generic, they provide electron spin echo envelope modulation (ESEEM) data that are rich with structural information derived from ligand hyperfine couplings. This chapter will focus on (2)H-ESEEM spectroscopy, an approach that we have taken for assigning these spectra and harvesting the unique information on Fe(II) coordination chemistry that they provide. While these spectroscopic measurements are routine, an emphasis will be placed on the analysis of cw-EPR and (2)H-ESEEM data using an unconstrained nonlinear optimization approach. These analysis methods are based on simple custom "scripts" that run in the MATLAB environment and that use EasySpin, a public-domain EPR simulation package, as their calculation engine. The examples provided here use a strategy that can be adapted for the treatment of most EPR measurements.

Aromatic amino acid hydroxylases are members of a larger group of enzymes that use a mononuclear nonheme Fe center to catalyze a variety of thermodynamically challenging reactions in which O2 is used in the oxidative transformation of substrates. The hydroxylase enzymes are catalytically active in the ferrous oxidation state and are high-spin. To render the catalytic site EPR-active, we have used nitric oxide (NO) as a surrogate for substrate O2 to form an S=3/2 paramagnetic center. While the continuous-wave (cw)-EPR spectra of NO-enzyme adducts are rather generic, they provide electron spin echo envelope modulation (ESEEM) data that are rich with structural information derived from ligand hyperfine couplings. This chapter will focus on (2)H-ESEEM spectroscopy, an approach that we have taken for assigning these spectra and harvesting the unique information on Fe(II) coordination chemistry that they provide. While these spectroscopic measurements are routine, an emphasis will be placed on the analysis of cw-EPR and (2)H-ESEEM data using an unconstrained nonlinear optimization approach. These analysis methods are based on simple custom "scripts" that run in the MATLAB environment and that use EasySpin, a public-domain EPR simulation package, as their calculation engine. The examples provided here use a strategy that can be adapted for the treatment of most EPR measurements.

The Nyika National Park, which spreads across northern Malawi into Zambia, is conventionally depicted as a ‘natural museum’, one of the last remaining untouched natural habitats in Africa. As this article demonstrates, however, from the late nineteenth century onwards the Nyika has been represented in a variety of different and often clashing forms involving local inhabitants (the Phoka) and European missionaries, explorers and administrators. From the late 1940s, a major influence shaping representations of the Nyika was Laurens van der Post, whose best-selling book, Venture to the Interior, provided a powerful image of the uncontaminated character of both the plateau and the Phoka people. At the same time, in his hitherto unpublished report on the Nyika, van der Post emphasised the potential of the plateau for European stock breeders and the inherently destructive nature of Phoka herders, gatherers and agriculturalists. The last section of the article focuses on the working out of van der Post's contrad...

Recent reports indicate that manganese (Mn), applied as a foliar fertilizer in tank mixtures with glyphosate, has the potential to antagonize glyphosate efficacy and reduce weed control. It was hypothesized that Mn2+ complexed with glyphosate in a similar manner to Ca2+, forming salts that were not readily absorbed and, thereby, reducing glyphosate efficacy. This study was conducted to confirm the interaction of Mn2+ and glyphosate and to measure the effect of Mn on glyphosate absorption and translocation in velvetleaf. In aqueous solutions, Mn2+ binds with solvent molecules and with chelating agents to form hexacoordinate complexes. The distribution of paramagnetic species, both the free manganous ion ([Mn{H2O}6]2+) and the Mn2+–glyphosate complex, in Mn–glyphosate solutions at various pH values were analyzed using electron paramagnetic resonance (EPR) spectroscopy. Glyphosate interaction with Mn appeared to increase as the pH was increased from spray solution levels (2.8 to 4.5) to levels commo...

To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.