Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605pRespiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mössbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mössbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ∼ 6 and g ∼ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ∼ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction

Genotype 1 hepatitis C virus envelope features that determine antiviral response assessed through optimal covariance networks

The poor response to the combined antiviral therapy of pegylated alfa-interferon and ribavarin for hepatitis C virus (HCV) infection may be linked to mutations in the viral envelope gene E1E2 (env), which can result in escape from the immune response and higher efficacy of viral entry. Mutations that result in failure of therapy most likely require compensatory mutations to achieve sufficient change in envelope structure and function. Compensatory mutations were investigated by determining positions in the E1E2 gene where amino acids (aa) covaried across groups of individuals. We assessed networks of covarying positions in E1E2 sequences that differentiated sustained virological response (SVR) from non-response (NR) in 43 genotype 1a (17 SVR), and 49 genotype 1b (25 SVR) chronically HCV-infected individuals. Binary integer programming over covariance networks was used to extract aa combinations that differed between response groups. Genotype 1a E1E2 sequences exhibited higher degrees of covariance and clustered into 3 main groups while 1b sequences exhibited no clustering. Between 5 and 9 aa pairs were required to separate SVR from NR in each genotype. aa in hypervariable region 1 were 6 times more likely than chance to occur in the optimal networks. The pair 531-626 (EI) appeared frequently in the optimal networks and was present in 6 of 9 NR in one of the 1a clusters. The most frequent pairs representing SVR were 431-481 (EE), 500-522 (QA) in 1a, and 407-434 (AQ) in 1b. Optimal networks based on covarying aa pairs in HCV envelope can indicate features that are associated with failure or success to antiviral therapy

The Functionality of the three-sited Ferroxidase center of E. coli Bacterial Ferritin (EcFtnA)

At least three ferritins are found in the bacterium Escherichia coli, the heme-containing bacterioferritin (EcBFR) and two non-heme bacterial ferritins (EcFtnA and EcFtnB). In addition to the conserved A- and B-sites of the diiron ferroxidase center, EcFtnA has a third iron-binding site (the C-site) of unknown function that is nearby the diiron site. In the present work, the complex chemistry of iron oxidation and deposition in EcFtnA has been further defined through a combination of oximetry, pH stat, stopped-flow and conventional kinetics, UV-visible, fluorescence and EPR spectroscopic measurements on the wildtype protein and site-directed variants of the A-, B- and C-sites. The data reveal that, while H2O2 is a product of dioxygen reduction in EcFtnA and oxidation occurs with a stoichiometry of Fe(II)/O2 ~ 3:1, most of the H2O2 produced is consumed in subsequent reactions with a 2:1 Fe(II)/H2O2 stoichiometry, thus suppressing hydroxyl radical formation. While the A- and B-sites are essential for rapid iron oxidation, the C-site slows oxidation and suppresses iron turnover at the ferroxidase center. A tyrosyl radical, assigned to Tyr24 near the ferroxidase center, is formed during iron oxidation and its possible significance to the function of the protein is discussed. Taken as a whole, the data indicate that there are multiple iron-oxidation pathways in EcFtnA with O2 and H2O2 as oxidants. Furthermore, the data are inconsistent with the C-site being a transit site, providing iron to the A- and B-sites, and does not support a universal mechanism for iron oxidation in all ferritins as recently proposed

Soil enzyme activities in the rhizosphere of field-grown sugar beet inoculated with the biocontrol agent Pseudomonas fluorescens F113

The original publication is available at www.springerlink.com . Copyright Springer DOI : 10.1007/s003740050397Pseudomonas fluorescens F113, which produces the antimicrobial compound 2,4-diacetylphloroglucinol is a prospective biocontrol agent. Soil enzyme activities were used to investigate the ecological impact of strain F113 in the rhizosphere of field-grown sugar beet. There were distinct trends in rhizosphere enzyme activities in relation to soil chemistry (studied by electro-ultrafiltration). The activities of enzymes from the phosphorus cycle (acid phosphatase, alkaline phosphatase and phosphodiesterase) and of arylsulphatase were negatively correlated with the amount of readily available P, whereas urease activity was positively correlated with the latter. Significant correlations between electro-ultrafiltration nutrient levels and enzyme activity in the rhizosphere were obtained, highlighting the usefulness of enzyme assays to document variations in soil nutrient cycling. Contrary to previous microcosm studies, which did not investigate plants grown to maturity, the biocontrol inoculant had no effect on enzyme activity or on soil chemistry in the rhizosphere. The results show the importance of homogenous soil microcosm systems, used in previous work, in risk assessment studies, where inherent soil variability is minimised, and where an effect of the pseudomonad on soil enzymology could be detected.Peer reviewe

Opposite Movement of the External Gate of a Glutamate Transporter Homolog upon Binding Cotransported Sodium Compared with Substrate

Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homolog Glt Ph . It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na + and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na + bound state is unknown. We here use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter Glt Ph from Pyrococcus horikoshi to examine conformational changes in HP2. Surprisingly, the cotransported substrates Na + and aspartate induce opposite movements of HP2. We find that in the apo state, HP2 is in a similar conformation as in the aspartate-bound closed state. Na + binding to the apo state opens HP2, whereas the subsequent binding of aspartate closes HP2. Our findings show that Na + binding opens and stabilizes the extracellular gate, thereby allowing for amino acid substrate binding. In contrast, in the absence of Na + and aspartate, HP2 closes, suggesting a potential mechanism for the translocation of the empty binding pocket necessary to complete the transport cycle. The finding that physiological Na + concentrations stabilize the open HP2 state would ensure that the outward-facing conformation of the transporter is maintained in physiological solutions and that glutamate transporters are ready to quickly bind glutamate released from glutamatergic synapses

Carboxylate as the Protonation Site in (Peroxo)diiron(III) Model Complexes of Soluble Methane Monooxygenase and Related Diiron Proteins

Dioxygen activation by carboxylate-bridged diiron enzymes is involved in essential biological processes ranging from DNA synthesis and hydrocarbon metabolism to cell proliferation.1-3 The carboxylate-bridged diiron superfamily of proteins includes ribonucleotide reductase (RNR),4 Δ9 desaturase,5 bacterial multicomponent monooxygenases (BMMs),6,7 and most recently human deoxyhypusine hydroxylase (hDOHH).3 In all of these systems, the O2 reduction step proceeds through a (peroxo)- diiron(III) intermediate in which the resulting peroxo ligand is proposed to bridge two iron atoms in a μ-1,2 or μ-η2η2 coordination mode.8-10 Extensive studies of soluble methane monooxygenase (sMMO), a BMM family member that oxidizes methane to methanol, reveal that the generation and activation of Fe2O2 units requires protons.11,12 Given the complexity of protein environments, identifying the sites involved in such proton translocation processes and their effect on O2 activation is not a trivial undertaking.National Institute of General Medical Sciences (U.S.) (grant GM032134)National Institute of General Medical Sciences (U.S.) (grant GM74785

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem

Versatile Reactivity of a Solvent-Coordinated Diiron(II) Compound: Synthesis and Dioxygen Reactivity of a Mixed-Valent Fe [superscript II] Fe [superscript III] Species

A new, DMF-coordinated, preorganized diiron compound [Fe[subscript 2](N-Et-HPTB)(DMF)[subscript 4]](BF[subscript 4])[subscript 3] (1) was synthesized, avoiding the formation of [Fe(N-Et-HPTB)](BF4)2 (10) and [Fe2(N-Et-HPTB)(μ-MeCONH)](BF[subscript 4])[subscript 2] (11), where N-Et-HPTB is the anion of N,N,N′,N′-tetrakis[2-(1-ethylbenzimidazolyl)]-2-hydroxy-1,3-diaminopropane. Compound 1 is a versatile reactant from which nine new compounds have been generated. Transformations include solvent exchange to yield [Fe[subscript 2](N-Et-HPTB)(MeCN)[subscript 4]](BF[subscript 4])[subscript 3] (2), substitution to afford [Fe[subscript 2](N-Et-HPTB)(μ-RCOO)](BF[subscript 4])[subscript 2] (3, R = Ph; 4, RCOO = 4-methyl-2,6-diphenyl benzoate]), one-electron oxidation by (Cp[subscript 2]Fe)(BF[subscript 4]) to yield a Robin–Day class II mixed-valent diiron(II,III) compound, [Fe[subscript 2](N-Et-HPTB)(μ-PhCOO)(DMF)[subscript 2]](BF[subscript 4])[subscript 3] (5), two-electron oxidation with tris(4-bromophenyl)aminium hexachloroantimonate to generate [Fe[subscript 2](N-Et-HPTB)Cl[subscript 3](DMF)](BF[subscript 4])[subscript 2] (6), reaction with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl to form [Fe[subscript 5](N-Et-HPTB)[subscript 2](μ-OH)[subscript 4](μ-O)(DMF)[subscript 2]](BF[subscript 4])[subscript 4] (7), and reaction with dioxygen to yield an unstable peroxo compound that decomposes at room temperature to generate [Fe[subscript 4](N-Et-HPTB)2(μ-O)[subscript 3](H[subscript 2]O)[subscript 2]](BF[subscript 4])·8DMF (8) and [Fe[subscript 4](N-Et-HPTB)[subscript 2](μ-O)[subscript 4]](BF[subscript 4])[subscript 2] (9). Compound 5 loses its bridging benzoate ligand upon further oxidation to form [Fe[subscript 2](N-Et-HPTB)(OH)[subscript 2](DMF)[subscript 2]](BF[subscript 4])[subscript 3] (12). Reaction of the diiron(II,III) compound 5 with dioxygen was studied in detail by spectroscopic methods. All compounds (1–12) were characterized by single-crystal X-ray structure determinations. Selected compounds and reaction intermediates were further examined by a combination of elemental analysis, electronic absorption spectroscopy, Mössbauer spectroscopy, EPR spectroscopy, resonance Raman spectroscopy, and cyclic voltammetry.National Institute of General Medical Sciences (U.S.) (Grant GM032134)Alexander von Humboldt-Stiftung (Postdoctoral Fellowship

Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize

International audienceAims: To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere. Methods and Results: Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 · 103 (for UAP-154) and 4 · 104 CFU g)1 (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 104 to 108 CFU g)1 soil. Conclusion: BOX-based SCAR markers were useful to find primers for strainspecific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Significance and Impact of the Study: Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535