Characterization of the free energy dependence of an interprotein electron transfer reaction by variation of pH and site-directed mutagenesis

AbstractThe interprotein electron transfer (ET) reactions of the cupredoxin amicyanin, which mediates ET from the tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase to cytochrome c-551i have been extensively studied. However, it was not possible to perform certain key experiments in that native system. This study examines the ET reaction from reduced amicyanin to an alternative electron acceptor, the diheme protein MauG. It was possible to vary the ΔG° for this ET reaction by simply changing pH to determine the dependence of kET on ΔG°. A P94A mutation of amicyanin significantly altered its oxidation–reduction midpoint potential value. It was not possible to study the ET from reduced P94A amicyanin to cytochrome c-551i in the native system because that reaction was kinetically coupled. However, the reaction from reduced P94A amicyanin to MauG was a true ET reaction and it was possible to determine values of reorganization energy (λ) and electronic coupling for the reactions of this variant as well as native amicyanin. Comparison of the λ values associated with the ET reactions between amicyanin and the TTQ of methylamine dehydrogenase, the diheme center of MauG and the single heme of cytochrome c-551i, provides insight into the factors that dictate the λ values for the respective reactions. These results demonstrate how study of ET reactions with alternative redox partner proteins can complement and enhance our understanding of the reactions with the natural redox partners, and further our understanding of mechanisms of protein ET reactions

Structures of MauG in complex with quinol and quinone MADH

MauG has been cocrystallized with methylamine dehydrogenase (MADH) with its TTQ cofactor in the o-quinol (TTQ(OQ)) and quinone (TTQ(OX)) forms and the structures of the resulting complexes have been solved. The TTQ(OQ) structure crystallized in either space group P2(1) or C2, while the TTQ(OX) structure crystallized in space group P1. The previously solved structure of MauG in complex with MADH bearing an incompletely formed TTQ cofactor (preMADH) also crystallized in space group P1, although with different unit-cell parameters. Despite the changes in crystal form, the structures are virtually identical, with only very minor changes at the protein-protein interface. The relevance of these structures with respect to the measured changes in affinity between MauG and various forms of MADH is discussed

Tryptophan-mediated charge-resonance stabilization in the bis-Fe(IV) redox state of MauG

The diheme enzyme MauG catalyzes posttranslational modifications of a methylamine dehydrogenase precursor protein to generate a tryptophan tryptophylquinone cofactor. The MauG-catalyzed reaction proceeds via a bis-Fe(IV) intermediate in which one heme is present as Fe(IV)=O and the other as Fe(IV) with axial histidine and tyrosine ligation. Herein, a unique near-infrared absorption feature exhibited specifically in bis-Fe(IV) MauG is described, and evidence is presented that it results from a charge-resonance-transition phenomenon. As the two hemes are physically separated by 14.5 angstrom, a hole-hopping mechanism is proposed in which a tryptophan residue located between the hemes is reversibly oxidized and reduced to increase the effective electronic coupling element and enhance the rate of reversible electron transfer between the hemes in bis-Fe(IV) MauG. Analysis of the MauG structure reveals that electron transfer via this mechanism is rapid enough to enable a charge-resonance stabilization of the bis-Fe(IV) state without direct contact between the hemes. The finding of the charge-resonance-transition phenomenon explains why the bis-Fe(IV) intermediate is stabilized in MauG and does not permanently oxidize its own aromatic residues

Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis

Despite the importance of tryptophan (Trp) radicals in biology, very few radicals have been trapped and characterized in a physiologically meaningful context. Here we demonstrate that the diheme enzyme MauG uses Trp radical chemistry to catalyze formation of a Trp-derived tryptophan tryptophylquinone cofactor on its substrate protein, premethylamine dehydrogenase. The unusual six-electron oxidation that results in tryptophan tryptophylquinone formation occurs in three discrete two-electron catalytic steps. Here the exact order of these oxidation steps in the processive six-electron biosynthetic reaction is determined, and reaction intermediates are structurally characterized. The intermediates observed in crystal structures are also verified in solution using mass spectrometry. Furthermore, an unprecedented Trp-derived diradical species on premethylamine dehydrogenase, which is an intermediate in the first two-electron step, is characterized using high-frequency and -field electron paramagnetic resonance spectroscopy and UV-visible absorbance spectroscopy. This work defines a unique mechanism for radical-mediated catalysis of a protein substrate, and has broad implications in the areas of applied biocatalysis and understanding of oxidative protein modification during oxidative stress

Enzymatic and Electron Transfer Activities in Crystalline Protein Complexes

Enzymatic and electron transfer activities have been studied by polarized absorption spectroscopy in single crystals of both binary and ternary complexes of methylamine dehydrogenase (MADH) with its redox partners. Within the crystals, MADH oxidizes methylamine, and the electrons are passed from the reduced tryptophan tryptophylquinone (TTQ) cofactor to the copper of amicyanin and to the heme of cytochrome c551i via amicyanin. The equilibrium distribution of electrons among the cofactors, and the rate of heme reduction after reaction with substrate, are both dependent on pH. The presence of copper in the ternary complex is not absolutely required for electron transfer from TTQ to heme, but its presence greatly enhances the rate of electron flow to the heme

Role of macrophage sialoadhesin in host defense against the sialylated pathogen group B <em>Streptococcus</em>

ABSTRACT: Several bacterial pathogens decorate their surfaces with sialic acid (Sia) residues within cell wall components or capsular exopolysaccharides. Sialic acid expression can promote bacterial virulence by blocking complement activation or by engagement of inhibitory sialic acid-binding immunoglobulin-like lectins (Siglecs) on host leukocytes. Expressed at high levels on splenic and lymph node macrophages, sialoadhesin (Sn) is a unique Siglec with an elongated structure that lacks intracellular signaling motifs. Sialoadhesin allows macrophage to engage certain sialylated pathogens and stimulate inflammatory responses, but the in vivo significance of sialoadhesin in infection has not been shown. We demonstrate that macrophages phagocytose the sialylated pathogen group B Streptococcus (GBS) and increase bactericidal activity via sialoadhesin-sialic-acid-mediated recognition. Sialoadhesin expression on marginal zone metallophillic macrophages in the spleen trapped circulating GBS and restricted the spread of the GBS to distant organs, reducing mortality. Specific IgM antibody responses to GBS challenge were also impaired in sialoadhesin-deficient mice. Thus, sialoadhesin represents a key bridge to orchestrate innate and adaptive immune defenses against invasive sialylated bacterial pathogens. KEY MESSAGE: Sialoadhesin is critical for macrophages to phagocytose and clear GBS. Increased GBS organ dissemination in the sialoadhesin-deficient mice. Reduced anti-GBS IgM production in the sialoadhesin-deficient mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00109-014-1157-y) contains supplementary material, which is available to authorized users

Column-integrated aerosol optical properties in Sodankylä (Finland) during the Solar Induced Fluorescence Experiment (SIFLEX-2002).

A study has been made of the column aerosols using solar irradiance extinction measurements at ground level in a boreal region (Sodankyla ̈, Finland) during spring 2002. The aerosol properties have been related to air mass origin. In general, the aerosol levels were observed to be very low, independent of the air mass origin, with an aerosol optical depth (AOD) value at 500 nm of less than 0.09 ± 0.03. Two characteristic patterns were observed depending on whether the air masses originated in the north and west or from the south and east. In the first case (north and west origins) the aerosol load was very small, with very low optical depths in the range 0.03 ± 0.02 to 0.09 ± 0.03 for 500 nm wavelengths. The size distribution usually showed two modes, with a strong contribution from the large-particle mode, probably a consequence of the presence of maritime-type particles originating in the ocean. When the air masses originated from a south or east direction, the behavior was the opposite to that noted before. In these cases the AOD was rather larger, above all for air masses originating in central Europe and Russia with an average value at 500 nm of 0.14 ± 0.02

Thermodynamics of mixing in diopside-jadeite, CaMgSi2O6-NaAlSi2O6, solid solution from staticlattice energy calculations

Static lattice energy calculations (SLEC), based on empirical interatomic potentials, have beenperformed for a set of 800 different structures in a 2 2 4 supercell of C2/c diopside with compositionsbetween diopside and jadeite, and with different states of order of the exchangeable Na/Ca and Mg/Al cations. Excess static energies of these structures have been cluster expanded in a basis set of 37 pair-interaction parameters. These parameters have been used to constrain Monte Carlo simulations of temperature-dependent properties in the range of 273?2,023 K and to calculate a temperature?composition phase diagram. The simulations predict the order?disorder transition in omphacite at1,150 20C in good agreement with the experimental data of Carpenter (Mineral Petrol 78:433?440, 1981). The stronger ordering of Mg/Al within the M1 site than of Ca/Na in the M2 site is attributed to the shorter M1?M1 nearest-neighbor distance, and, consequently, the stronger ordering force. The comparison of the simulated relationship between the order parameters corresponding to M1 and M2 sites with the X-ray refinement data on natural omphacites (Boffa Ballaran et al. in Am Mineral83:419?433, 1998) suggests that the cation ordering becomes kinetically ineffective at about 600C

Membrane Damage Elicits an Immunomodulatory Program in Staphylococcus aureus

The Staphylococcus aureus HrtAB system is a hemin-regulated ABC transporter composed of an ATPase (HrtA) and a permease (HrtB) that protect S. aureus against hemin toxicity. S. aureus strains lacking hrtA exhibit liver-specific hyper-virulence and upon hemin exposure over-express and secrete immunomodulatory factors that interfere with neutrophil recruitment to the site of infection. It has been proposed that heme accumulation in strains lacking hrtAB is the signal which triggers S. aureus to elaborate this anti-neutrophil response. However, we report here that S. aureus strains expressing catalytically inactive HrtA do not elaborate the same secreted protein profile. This result indicates that the physical absence of HrtA is responsible for the increased expression of immunomodulatory factors, whereas deficiencies in the ATPase activity of HrtA do not contribute to this process. Furthermore, HrtB expression in strains lacking hrtA decreases membrane integrity consistent with dysregulated permease function. Based on these findings, we propose a model whereby hemin-mediated over-expression of HrtB in the absence of HrtA damages the staphylococcal membrane through pore formation. In turn, S. aureus senses this membrane damage, triggering the increased expression of immunomodulatory factors. In support of this model, wildtype S. aureus treated with anti-staphylococcal channel-forming peptides produce a secreted protein profile that mimics the effect of treating ΔhrtA with hemin. These results suggest that S. aureus senses membrane damage and elaborates a gene expression program that protects the organism from the innate immune response of the host

Effective health care for older people living and dying in care homes: A realist review

Background: Care home residents in England have variable access to health care services. There is currently no coherent policy or consensus about the best arrangements to meet these needs. The purpose of this review was to explore the evidence for how different service delivery models for care home residents support and/or improve wellbeing and health-related outcomes in older people living and dying in care homes. Methods: We conceptualised models of health care provision to care homes as complex interventions. We used a realist review approach to develop a preliminary understanding of what supported good health care provision to care homes. We completed a scoping of the literature and interviewed National Health Service and Local Authority commissioners, providers of services to care homes, representatives from the Regulator, care home managers, residents and their families. We used these data to develop theoretical propositions to be tested in the literature to explain why an intervention may be effective in some situations and not others. We searched electronic databases and related grey literature. Finally the findings were reviewed with an external advisory group. Results: Strategies that support and sustain relational working between care home staff and visiting health care professionals explained the observed differences in how health care interventions were accepted and embedded into care home practice. Actions that encouraged visiting health care professionals and care home staff jointly to identify, plan and implement care home appropriate protocols for care, when supported by ongoing facilitation from visiting clinicians, were important. Contextual factors such as financial incentives or sanctions, agreed protocols, clinical expertise and structured approaches to assessment and care planning could support relational working to occur, but of themselves appeared insufficient to achieve change. Conclusion: How relational working is structured between health and care home staff is key to whether health service interventions achieve health related outcomes for residents and their respective organisations. The belief that either paying clinicians to do more in care homes and/or investing in training of care home staff is sufficient for better outcomes was not supported.This research was funded by National Institute of Health Research Health Service Delivery and Research programme (HSDR 11/021/02)