The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core.

Ferritins are multimeric cage-forming proteins that play a crucial role in cellular iron homeostasis. All H-chain-type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α-helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron-release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme-ferroxidase centre electron transfer pathway.

Implementation of Exercise Management Services Among Sports Medicine Physicians in the United States.

OBJECTIVE: Assessment of physical activity and exercise prescription has been widely supported by many organizations, yet provision of such services remains limited in the United States. We sought to uncover why such services have not been widely adopted. DESIGN: The American Medical Society for Sports Medicine organized a task force to canvas physicians and survey the American Medical Society for Sports Medicine membership. SETTING: Peer-to-peer and telecommunication discussions and web-based questionnaires. PARTICIPANTS: Sports medicine physicians in the United States. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Percentage of sports medicine physicians who provide exercise management services and mechanisms of billing for exercise management, identify barriers to such services, and identify industry collaborations for promoting physical activity through physicians. RESULTS: Three of 4 sports medicine physicians spend at least 1 min encouraging exercise with patients, using Evaluation and Management codes to bill or receive credit. Exercise counseling is often bundled within other patient care. Few health plans leverage the patient's relationship with a primary care physician to promote exercise. Most employed sports medicine physicians do not receive incentives to incorporate exercise counseling into practice, and only 1 in 6 have decision-making authority to hire an exercise professional. Major obstacles are the lack of a business model and knowledge about exercise prescription. CONCLUSION: The existing E&M codes adequately characterize the work, but physicians desire greater payment or credit for providing exercise management services. Physicians desire to do more exercise prescription, but health system bureaucracy, inadequate support, and economic disincentives are barriers to the provision of exercise management services.

Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we use single-cell RNA sequencing (scRNA-seq) to examine immune cells in patient and control cohorts. Postexertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. To detect changes coincident with PEM, we applied scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients display classical monocyte dysregulation suggestive of inappropriate differentiation and migration to tissue. We identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlates with disease severity. Comparing the transcriptome at baseline and postexercise challenge, we discover patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation in platelets.

Recovery from Exercise in Persons with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Background and Objectives: Post-exertional malaise (PEM) is the hallmark of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), but there has been little effort to quantitate the duration of PEM symptoms following a known exertional stressor. Using a Symptom Severity Scale (SSS) that includes nine common symptoms of ME/CFS, we sought to characterize the duration and severity of PEM symptoms following two cardiopulmonary exercise tests separated by 24 h (2-day CPET). Materials and Methods: Eighty persons with ME/CFS and 64 controls (CTL) underwent a 2-day CPET. ME/CFS subjects met the Canadian Clinical Criteria for diagnosis of ME/CFS; controls were healthy but not participating in regular physical activity. All subjects who met maximal effort criteria on both CPETs were included. SSS scores were obtained at baseline, immediately prior to both CPETs, the day after the second CPET, and every two days after the CPET-1 for 10 days. Results: There was a highly significant difference in judged recovery time (ME/CFS = 12.7 ± 1.2 d; CTL = 2.1 ± 0.2 d, mean ± s.e.m., Chi2 = 90.1, p < 0.0001). The range of ME/CFS patient recovery was 1-64 days, while the range in CTL was 1-10 days; one subject with ME/CFS had not recovered after one year and was not included in the analysis. Less than 10% of subjects with ME/CFS took more than three weeks to recover. There was no difference in recovery time based on the level of pre-test symptoms prior to CPET-1 (F = 1.12, p = 0.33). Mean SSS scores at baseline were significantly higher than at pre-CPET-1 (5.70 ± 0.16 vs. 4.02 ± 0.18, p < 0.0001). Pharmacokinetic models showed an extremely prolonged decay of the PEM response (Chi2 > 22, p < 0.0001) to the 2-day CPET. Conclusions: ME/CFS subjects took an average of about two weeks to recover from a 2-day CPET, whereas sedentary controls needed only two days. These data quantitate the prolonged recovery time in ME/CFS and improve the ability to obtain well-informed consent prior to doing exercise testing in persons with ME/CFS. Quantitative monitoring of PEM symptoms may provide a method to help manage PEM.

Protein encapsulation within the internal cavity of a bacterioferritin.

The thermal and chemical stability of 24mer ferritins has led to attempts to exploit their naturally occurring nanoscale (8 nm) internal cavities for biotechnological applications. An area of increasing interest is the encapsulation of molecules either for medical or biocatalysis applications. Encapsulation requires ferritin dissociation, typically induced using high temperature or acidic conditions (pH ≥ 2), which generally precludes the inclusion of fragile cargo such as proteins or peptide fragments. Here we demonstrate that minimizing salt concentration combined with adjusting the pH to ≤8.5 (i.e. low proton/metal ion concentration) reversibly shifts the naturally occurring equilibrium between dimeric and 24meric assemblies of Escherichia coli bacterioferritin (Bfr) in favour of the disassembled form. Interconversion between the different oligomeric forms of Bfr is sufficiently slow under these conditions to allow the use of size exclusion chromatography to obtain wild type protein in the purely dimeric and 24meric forms. This control over association state was exploited to bind heme at natural sites that are not accessible in the assembled protein. The potential for biotechnological applications was demonstrated by the encapsulation of a small, acidic [3Fe-4S] cluster-containing ferredoxin within the Bfr internal cavity. The capture of ∼4-6 negatively charged ferredoxin molecules per cage indicates that charge complementarity with the inner protein surface is not an essential determinant of successful encapsulation.

Plasma metabolomics reveals disrupted response and recovery following maximal exercise in myalgic encephalomyelitis/chronic fatigue syndrome.

Post-exertional malaise (PEM) is a hallmark symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We monitored the evolution of 1157 plasma metabolites in 60 ME/CFS (45 female, 15 male) and 45 matched healthy control participants (30 female, 15 male) before and after 2 maximal cardiopulmonary exercise test (CPET) challenges separated by 24 hours, with the intent of provoking PEM in patients. Four time points allowed exploration of the metabolic response to maximal energy-producing capacity and the recovery pattern of participants with ME/CFS compared with the healthy control group. Baseline comparison identified several significantly different metabolites, along with an enriched percentage of yet-to-be identified compounds. Additionally, temporal measures demonstrated an increased metabolic disparity between cohorts, including unknown metabolites. The effects of exertion in the ME/CFS cohort predominantly highlighted lipid-related as well as energy-related pathways and chemical structure clusters, which were disparately affected by the first and second exercise sessions. The 24-hour recovery period was distinct in the ME/CFS cohort, with over a quarter of the identified pathways statistically different from the controls. The pathways that are uniquely different 24 hours after an exercise challenge provide clues to metabolic disruptions that lead to PEM. Numerous altered pathways were observed to depend on glutamate metabolism, a crucial component of the homeostasis of many organs in the body, including the brain.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2 O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+ , was exposed to O2 or H2 O2 . We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2 O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O2 . Initially formed Fe3+ can further react with H2 O2 (producing protein bound radicals) but relaxes within seconds to an H2 O2 -unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2 O2 rather than sequester iron.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Evolution of the sheep coat: the impact of domestication on its structure and development.

Wild sheep and many primitive domesticated breeds have two coats: coarse hairs covering shorter, finer fibres. Both are shed annually. Exploitation of wool for apparel in the Bronze Age encouraged breeding for denser fleeces and continuously growing white fibres. The Merino is regarded as the culmination of this process. Archaeological discoveries, ancient images and parchment records portray this as an evolutionary progression, spanning millennia. However, examination of the fleeces from feral, two-coated and woolled sheep has revealed a ready facility of the follicle population to change from shedding to continuous growth and to revert from domesticated to primitive states. Modifications to coat structure, colour and composition have occurred in timeframes and to sheep population sizes that exclude the likelihood of variations arising from mutations and natural selection. The features are characteristic of the domestication phenotype: an assemblage of developmental, physiological, skeletal and hormonal modifications common to a wide variety of species under human control. The phenotypic similarities appeared to result from an accumulation of cryptic genetic changes early during vertebrate evolution. Because they did not affect fitness in the wild, the mutations were protected from adverse selection, becoming apparent only after exposure to a domestic environment. The neural crest, a transient embryonic cell population unique to vertebrates, has been implicated in the manifestations of the domesticated phenotype. This hypothesis is discussed with reference to the development of the wool follicle population and the particular roles of Notch pathway genes, culminating in the specific cell interactions that typify follicle initiation.

Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn.

Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres.

Bacterial iron detoxification at the molecular level.

Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable "free" iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.

Comparison of the structural dynamic and mitochondrial electron-transfer properties of the proapoptotic human cytochrome c variants, G41S, Y48H and A51V.

Mitochondrial cytochrome c is associated with electron transfer in the respiratory chain and in apoptosis. Four cytochrome c variants have been identified in families that suffer from mild autosomal dominant thrombocytopenia, a platelet disorder associated with increased apoptosis. Three out of the four substitutions, G41S, Y48H and A51V are located on the 40-57 Ω-loop. The G41S and Y48H variants perturb key physicochemical and dynamic properties that result in enhanced functional features associated with apoptotic activity. Herein we characterise the ferric A51V variant. We show by chemical denaturation that this variant causes the native state to be destabilized. Through azide binding kinetics, the population of a pentacoordinate heme form, whereby the Met80 axial ligand is dissociated, is estimated to be of equal magnitude to that found in the Y48H variant. This pentacoordinate form gives rise to peroxidase activity, which despite the similar pentacoordinate population of the A51V variant to that of the Y48H variant, the peroxidase activity of the A51V variant is suppressed. Far-UV circular dichroism spectroscopy and pH jump studies, suggest that a combination of structural and dynamic features in addition to the population of the pentacoordinate form regulate peroxidase activity in these disease variants. Additionally, the steady-state ratio of ferric/ferrous cytochrome c when in turnover with cytochrome c oxidase has been investigated for all 40-57 Ω-loop variants. These studies show that the lower pKa of the alkaline transition for the disease causing variants increases the ferric to ferrous heme ratio, indicating a possible influence on respiration in vivo.

Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A.

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P's structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.

Reaction of O2 with a diiron protein generates a mixed-valent Fe2+/Fe3+ center and peroxide.

The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron-O2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron-O2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

Naturally Occurring Disease-Related Mutations in the 40-57 Ω-Loop of Human Cytochrome c Control Triggering of the Alkaline Isomerization.

Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71-85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (p Ktrigger). A second deprotonation stabilizes a pentacoordinate heme form (p Ka2). In this study, site-directed variants at the 41 and 48 positions in the 40-57 Ω-loop and at the 81 and 83 positions in the 71-85 Ω-loop reveal that conformational transitions in the 71-85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40-57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the p Ktrigger and increase p Ka2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80-Fe(III) bond strength.

Emissions mitigation opportunities for savanna countries from early dry season fire management.

Savanna fires produce significant emissions globally, but if managed effectively could provide an important mitigation opportunity, particularly in African least developed countries. Here we show global opportunities for emissions reductions through early dry season burning for 37 countries including: 29 countries in Africa (69.1 MtCO2-e yr-1), six countries in South America (13.3 MtCO2-e yr-1), and Australia and Papua New Guinea (6.9 MtCO2-e yr-1). Emissions reduction estimates are based on the successful approach developed in Australia to reduce emissions from savanna fires using global-scale, remotely sensed estimates of monthly emissions. Importantly, 20 least developed countries in Africa account for 74% of the mitigation potential (60.2 MtCO2-e yr-1). More than 1.02 million km2 of savanna dominated protected areas within these countries could be used as pilot sites to test and advance a regional approach to mitigation efforts for savanna fires in Africa. Potential versus actual abatement opportunities are discussed.

The N-terminal domains of Bacillus subtilis CopA do not form a stable complex in the absence of their inter-domain linker.

Copper-transporting P-type ATPases, which play important roles in trafficking Cu(I) across membranes for the biogenesis of copper proteins or for copper detoxification, contain a variable number of soluble metal-binding domains at their N-termini. It is increasingly apparent that these play an important role in regulating copper transport in a Cu(I)-responsive manner, but how they do this is unknown. CopA, a Cu(I)-transporter from Bacillus subtilis, contains two N-terminal soluble domains that are closely packed, with inter-domain interactions at two principal regions. Here, we sought to determine the extent to which the domains interact in the absence of their inter-domain covalent linker, and how their Cu(I)-binding properties are affected. Studies of a 1:1 mixture of separate CopAa and CopAb domains showed that the domains do not form a stable complex, with only indirect evidence of a weak interaction between them. Their Cu(I)-binding behaviour was distinct from that of the two domain protein and consistent with a lack of interaction between the domains. Cu(I)-mediated protein association was observed, but this occurred only between domains of the same type. Thus, the inter-domain covalent link between CopAa and CopAb is essential for inter-domain interactions and for Cu(I)-binding behaviour.