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1.
《Gene》1996,174(2):195-202
In a previous work, all three components of Comamonas testosteroni B-356 biphenyl (BPH)/chlorobiphenyls (PCBs) dioxygenase (dox) have been purified and characterized. They include an iron-sulphur protein (ISPBPH) which is the terminal oxygenase composed of two subunits (encoded by bphA and bphE), a ferredoxin (FERBPH) encoded by bphF and a reductase (REDBPH) encoded by bphG. bphG Is not located in the neighbourhood of bphAEF in B-356. We are reporting the cloning of B-356-bphG and the sequencing of B-356-BPH dox genes. Comparative analysis of the genes provided genetic evidence showing that two BPH dox lineages have emerged in Gram-negative bacteria. The main features of the lineage that includes B-356 are the location of bphG outside the bph gene cluster and the structure of REDBPH which is very distinct from all other aryl dioxygenase-reductases. 相似文献
2.
Alexander F. Arendsen Marcel de Vocht Yvonne B. M. Bulsink W. R. Hagen 《Journal of biological inorganic chemistry》1996,1(4):292-296
Aldehyde:ferredoxin oxidoreductase (AOR) from the hyperthermophilic archaeon Pyrococcus furiosus is a homodimeric protein. Each subunit carries one [4Fe-4S] cubane and a novel tungsten cofactor containing two pterins.
A single iron atom bridges between the subunits. AOR has previously been studied with EPR spectroscopy in an inactive form
known as the red tungsten protein (RTP): reduced RTP exhibits complex EPR interaction signals. We have now investigated the
active enzyme AOR with EPR, and we have found an S = 1/2 plus S = 3/2 spin mixture from a non-interacting [4Fe-4S]1+ cluster in the reduced enzyme. Oxidized AOR affords EPR signals typical for W(V) with g–values of 1.982, 1.953, and 1.885. The W(V) signals disappear at a reduction potential E
m,7.5 of +180 mV. This unexpectedly high value indicates that the active-site redox chemistry is based on the pterin part of the
cofactor.
Received: 18 December 1995 / Accepted: 26 March 1996 相似文献
3.
R. M. Davydov Joanne Smieja S. A. Dikanov Y. Zang Lawrence Que Jr. M. K. Bowman 《Journal of biological inorganic chemistry》1999,4(3):292-301
Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically
stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent
species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent
EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g
z
–g
av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized
by large g-anisotropy (g
z
–g
av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in
the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished
from each other and can help characterize the structure of mixed-valent centers in proteins.
Received: 27 June 1998 / Accepted: 25 February 1999 相似文献
4.
Ish K. Dhawan Roopali Roy Brian P. Koehler Swaranalatha Mukund Michael W. W. Adams Michael K. Johnson 《Journal of biological inorganic chemistry》2000,5(3):313-327
Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD)
spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (E
m=−368 mV) that undergoes redox cycling between an oxidized form with an S=0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S=3/2 (g=5.4; E/D=0.33) and S=1/2 (g=2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of
W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed
upon oxidation, termed the “low-potential” W(V) species (g=1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/W(V) and
W(V)/W(VI) states at physiologically relevant potentials (E
m=−335 and −280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g=1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of −34 mV and persists
up to at least +300 mV. At potentials above 0 mV, a major “high-potential” W(V) signal, g=1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples
of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox
properties of the sulfide-activated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential
species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E
m=−265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; E
m=+65 mV). Taken together, the W(V) species in sulfide-activated samples of Tl FOR maximally account for only 15% of the total
W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential
species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for
each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts
of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible
for the catalytic activity in sulfide-activated samples of Tl FOR.
Received: 9 September 1999 / Accepted: 17 February 2000 相似文献
5.
Andreas Erkens Klaus Schneider A. Müller 《Journal of biological inorganic chemistry》1996,1(2):99-110
In this study we confirmed the previous observation that the cytoplasmic NAD-linked hydrogenase of Alcaligenes eutrophus H16 is EPR-silent in the oxidized state. We also demonstrated the presence of significant Ni-EPR signals when the enzyme
was either reduced with the natural electron carrier NADH (5–10 mM) or carefully titrated with sodium dithionite to an intermediate,
narrow redox potential range (–280 to –350 mV). Reduction with NADH under argon atmosphere led to a complex EPR spectrum at
80 K with g values at 2.28, 2.20, 2.14, 2.10, 2.05, 2.01 and 2.00. This spectrum could be differentiated by special light/dark treatments
into three distinct signals: (1) the "classical" Ni-C signal with g values at 2.20, 2.14 and 2.01, observed with many hydrogenases in the reduced, active state; (2) the light-induced signal
(Ni-L) with g values at 2.28, 2.10 and 2.05 and (3) a flavin radical (FMN semiquinone) signal at g = 2.00. The assignment of the Ni-EPR signal was clearly confirmed by EPR spectra of hydrogenase labeled with 61Ni (nuclear spin I = 3/2) yielding a broadening of the Ni spectra at all g values and a resolved 61Ni hyperfine splitting into four lines of the low field edge in the case of the light-induced Ni-EPR signal. The redox potentials
determined at pH 7.0 for the described redox components were: for FMN –170 mV (midpoint potential, Em, for appearance), –200 mV (EPR signal intensity maximum) and –230 mV (Em for disappearance); for the Ni centre (Ni-C), –290 mV (Em for appearance), –305 mV (signal intensity maximum) and –325 mV (Em for disappearance). Exposure of the NADH-reduced hydrogenase to carbon monoxide led to an apparent Ni-CO species indicated
by a novel rhombic EPR signal with g values at 2.35, 2.08 and 2.01.
Received: 19 July 1995 / Accepted: 10 September 1995 相似文献
6.
Yean-Sung Jung I. R. Vassiliev J. H. Golbeck 《Journal of biological inorganic chemistry》1997,2(2):209-217
PsaC is a tightly bound ferredoxin in the Photosystem I (PS I) reaction center which contains two [4Fe-4S] clusters named
FA and FB. We recently proposed that the mixed-ligand FB cluster in C14DPsaC and the mixed-ligand FA cluster in C51DPsaC exist in a spin state of S=3/2, and that a spin state crossover to S=1/2 occurs when the PsaC mutants are rebound onto P700-FX cores. Since EPR signals from a highly rhombic S=3/2 spin state can be difficult to study, wild-type PsaC was reconstituted with iron and selenium to introduce an easily
detected S=7/2 spin state similar to that shown for Clostridial ferredoxin. When the unbound [4Fe-4Se] PsaC was chemically reduced, a sharp derivative resonance was found at g=5.171 attributed to the excited ±3/2 doublet from an S=7/2 spin multiplet. An additional peak was found at g=5.616 attributed to the superimposed ±1/2 and ±3/2 doublets from a highly rhombic S=3/2 spin multiplet, and an axial set of resonances found around g=2.0 attributed, in part, to a classical S=1/2 spin state. When the [4Fe-4Se] PsaC was rebound onto P700-FX cores, the spin population derived from the S=7/2 and 3/2 spin states was negligible. Illumination of the rebuilt PS I complex at 15 K resulted in two rhombic sets of
resonances, one with g values of 2.043, 1.941 and 1.854, diagnostic of FA, and the other with g values of 2.067, 1.941 and 1.878, diagnostic of FB. Chemical reduction with sodium dithionite at pH 10.5 or photoaccumulation by freezing during illumination resulted in a
set of resonances with g values of 2.046, 1.938, 1.920 and 1.883, characteristic of a spin-coupled FA
–/FB
– pair. The spin state crossover in this iron chalcogenide cluster is the first known to be induced by protein-protein association
and reinforces the hypothesis that an S=3/2 to 1/2 crossover occurs in the PS I-rebound mutants C14DPsaC and C51DPsaC.
Received: 6 August 1996 / Accepted: 28 December 1996 相似文献
7.
Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp 总被引:2,自引:0,他引:2
G. M. Gübitz D. Haltrich B. Latal W. Steiner 《Applied microbiology and biotechnology》1997,47(6):658-662
Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding
sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal
and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers).
Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others
acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released
(K
v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from
Penicillium simplicissimum (K
v = 0.15 l mPa−1s−1g−1) and a mannanase from Sclerotium rolfsii (K
v = 0.12 l mPa−1s−1g−1) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation
of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO
respectively. A less significant brightness increase was obtained with enzymes showing lower K
v values, such as a xylanase from Schizophyllum commune (Kv = 0.051 l mPa−1s−1g−1, 0.2% ISO) and a bacterial mannanase (K
v = 0.061 l mPa−1s−1g−1,0.5% ISO).
Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997 相似文献
8.
Involvement of the Terminal Oxygenase β Subunit in the Biphenyl Dioxygenase Reactivity Pattern toward Chlorobiphenyls 下载免费PDF全文
Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (α and β) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3′-dichlorobiphenyl over the double-para-substituted congener 4,4′-dichlorobiphenyl or the double-ortho-substituted congener 2,2′-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2′-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2′,5,5′-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH α or β subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the α subunit of ISPBPH of strain LB400 and the β subunit of ISPBPH of strain B-356 (the αLB400βB-356 chimera) and the αB-356βLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH β subunit influences BPH dox’s reactivity pattern toward chlorobiphenyls. Thus, if the α subunit were the sole determinant of the enzyme reactivity pattern, the αB-356βLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the αLB400βB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2′- and 3,3′-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl. 相似文献
9.
R. M. Davydov Stéphane Ménage M. Fontecave Astrid Gräslund A. Ehrenberg 《Journal of biological inorganic chemistry》1997,2(2):242-255
Several dinuclear diferric complexes were prepared, bridged by only an oxo ion, by an oxo ion and additionally one or two
carboxylate groups, or by two hydroxo ions. Frozen solutions of these EPR-silent diferric complexes were radiolytically reduced
by γ-irradiation at 77 K. The reduced forms gave EPR spectra typical of mixed-valent diiron species with S = 1/2. The yield was about 50% at high doses. With 57Fe replacing 56Fe the spectral features were broadened because of hyperfine interaction with one or both of the 57Fe nuclei. All oxo-bridged complexes give mixed-valent species with unbroadened EPR spectra observable up to 110 K. The anisotropy
is small, Δg = g
1–g
3<0.07, and g
av = (g
1+g
2+g
3)/3 falls in the range 1.93–1.96. Annealing at T≥115 K causes the EPR spectra to change and finally become more anisotropic, 0.14<Δg<0.32 and to have smaller g
av, 1.78<g
av<1.89. These spectra are observable only at T≤35 K. Radiolytic reduction of the diferric complexes with hydroxo bridges leads directly without annealing to this second
type of mixed-valent species. The annealing-induced transition from a primary to a secondary mixed-valent species in the case
of an oxo-bridged diferric starting complex is suggested to be a structural relaxation including protonation of the bridge.
Annealing at temperatures above ca. 180 K and final thawing causes the mixed-valent dinuclear species to disappear, probably
by disintegration to mononuclear species. These mixed-valent species are useful models for similar species in proteins, where
primary-type species may take part in transient electron transfer, and the secondary-type species are usually more persistent
forms in redox reactions. For proteins, available data show that there is a tendency for both primary and secondary species
to have larger g anisotropy, Δg, and smaller g
av, than obtained with the models. These differences suggest that the diiron centers of proteins are affected by their specific
surroundings. Our results also show that the nature of the bridge, oxo or hydroxo, may not always safely be determined from
a measured J value. Measurement of the EPR parameters, Δg, g
av and limiting temperature of observation (relaxation properties) of a mixed-valent complex, or, in the case of a diferric
complex, of the primary mixed-valent species induced by radiolysis at 77 K, offers an alternative empirically based method
to determine the nature of the bridge.
Received: 8 May 1996 / Accepted: 22 January 1997 相似文献
10.
The chromosomes of 31 species of Passiflora, distributed throughout the subgenera Astrophea, Calopathanthus, Distephana, Dysosmia, Passiflora, Plectostemma and Tacsonia were analysed. Three different karyotypes were observed: 2n = 12, 24, 36; 2n = 18, 72 and 2n = 20. The karyotype of these
species was almost always constituted of metacentric and submetacentric chromosomes with variable karyotype symmetry. In the
group with x = 6, represented by the subgenus Plectostemma, six diploid species with 2n = 12, one tetraploid with 2n = 24 (P. suberosa) and an intraspecific polyploid with 2n = 12, 36 (P. misera) were analysed. P. pentagona (subgenus Astrophea) may also be included in this karyological group since it presents 2n = 24 and may be of polyploid origin, with x = 6. The
interphase nuclei in this group were areticulate, except those of P. morifolia and P. pentagona with semi-reticulate characteristics. Two small terminal heterochromatic blocks, positive for chromomycin A3, were identified in the largest chromosome pair of P. capsularis and P. rubra, species very closely related, while P. tricuspis displayed four chromosomes with proximal blocks. In the group with x = 9, represented mainly by subgenus Passiflora, 20 species with 2n = 18 and one with 2n = 72 were studied. They presented chromosomes larger than those species with x = 6
and interphase nuclei of semi-reticulate type, except for P. mixta with areticulate nuclei. Four terminal CMA+ blocks were observed in P. edulis, six blocks in P. caerulea and P. racemosa, while five blocks were observed in the single P. amethystina plant analysed. P. foetida (subgenus Dysosmia), the only species with 2n = 20, exhibited six chromosomes with CMA+ blocks and interphase nuclei of the areticulate type. The meiotic analysis of representatives of the three groups (P. foetida, P. suberosa, P. cincinnata and P. racemosa) always presented regular pairing and regular chromosome segregation, except in P. jilekii where a tetravalent was observed. The analysis of the chromosome variation within the genus and the family suggests that
the base number of Passiflora may be x1 = 6 or x1 = 12, whereas x2 = 9 is only an important secondary base number.
Received April 11, 2000 Accepted October 5, 2000 相似文献
11.
Gil Rodas Mar Calvo Asunción Estruch Eduardo Garrido Guadalupe Ercilla Antoni Arcas Ramon Segura Josep L. Ventura 《European journal of applied physiology and occupational physiology》1998,77(6):511-516
Running economy (RE), defined as the steady-state of oxygen uptake (V˙O2) for a given running velocity, is a factor of sports performance the genetic component of which has seldom been reported
to date. We studied this component using a heritability index (HI) in a group of 32 male twins, 8 monozygotic (MZ) and 8 dizygotic
(DZ) pairs, all sportsmen with similar perinatal and environmental backgrounds. Zygocity was determined by the identity of
erythrocytic antigenic, protein and enzymatic polymorphism, and human leucocyte antigen serologic types between co-twins.
The subjects exercised twice on a treadmill, once until exhaustion and again at submaximal intensities. Pulmonary gas exchange
was measured continuously using an automatic analyser system during both tests. Blood samples were obtained during the recovery
period to determine lactate concentrations. No significant differences were observed between MZ and DZ, in respect of RE at
any speed or in maximal V˙O2 relative to body mass. Nevertheless, significant HI (P < 0.05) was found in maximal lactate concentrations (HI = 0.75) and in respiratory equivalent for oxygen at two speeds, 7 km · h−1 (HI = 0.71) and 8 km · h−1 (HI = 0.79), differences which probably suggest that there are differences in RE. In conclusion, we did not detect a genetic
component in RE or in maximal oxygen uptake, but a genetic component for markers of anaerobic metabolism was present.
Accepted: 5 November 1997 相似文献
12.
Eric Mandrou Paulo Ricardo Gherardi Hein Emilie Villar Philippe Vigneron Christophe Plomion Jean-Marc Gion 《Tree Genetics & Genomes》2012,8(2):353-364
Lignin content and composition are considered as mandatory traits of eucalyptus breeding programs, especially for pulp, paper,
and bioenergy production. In this article, we used 33 Eucalyptus urophylla full-sib families of an 8 × 8 factorial design to provide estimates of genetic parameters for lignin- and growth-related
traits. Secondly, from the sequencing of the 16 unrelated founders, we described the nucleotide and haplotype variability
of cinnamoyl-CoA reductase (CCR), a candidate gene for lignin-related traits encoding the cinnamoyl-CoA reductase. Finally, we tested the association between
CCR polymorphisms and trait variation using a mixed linear model. A high value of narrow sense heritability was obtained for
lignin content (h2 = 0.85) and S/G ratio (h2 = 0.62) indicating that these traits are under strong genetic control. High levels of nucleotide (θπ = 0.0131) and haplotype (Hd = 0.958) diversity were detected for CCR. From an initial set of 152 biallelic single nucleotide polymorphisms (SNPs), a subset of 65 nonredundant loci was selected.
Three intronic SNPs were found to be associated to the variation of S/G ratio after multiple testing correction. In the line
of what has been obtained in forest trees, these SNPs explained between 2.45% and 2.87% of the genetic variance of the trait.
This study demonstrates the interest of the candidate gene approach for quantitative trait nucleotide detection in Eucalyptus and paves the way to gene assisted selection of lignin composition in E. urophylla. 相似文献
13.
Hirano S Haruki M Takano K Imanaka T Morikawa M Kanaya S 《Applied microbiology and biotechnology》2006,69(6):672-681
Xanthobacter polyaromaticivorans sp. nov. 127W is a bacterial strain that is capable of degrading a wide range of cyclic aromatic compounds such as dibenzothiophene,
biphenyl, naphthalene, anthracene, and phenanthrene even under extremely low oxygen [dissolved oxygen (DO)≤0.2 ppm] conditions
(Hirano et al., Biosci Biotechnol Biochem 68:557–564, 2004). A major protein fraction carrying dibenzothiophene degradation
activity was purified. Based on its partial amino acid sequences, dbdCa gene encoding alpha subunit terminal oxygenase (DbdCa) and its flanking region were cloned and sequenced. A phylogenetic
analysis based on the amino acid sequence demonstrates that DbdCa is a member of a terminal oxygenase component of group IV
ring-hydroxylating dioxygenases for biphenyls and monocyclic aromatic hydrocarbons, rather than group III dioxygenases for
polycyclic aromatic hydrocarbons. Gene disruption in dbdCa abolished almost of the degradation activity against biphenyl, dibenzothiophene, and anthracene. The gene disruption also
impaired degradation activity of the strain under extremely low oxygen conditions (DO≤0.2 ppm). These results indicate that
Dbd from 127W represents a group IV dioxygenase that is functional even under extremely low oxygen conditions. 相似文献
14.
Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins 总被引:59,自引:33,他引:26 下载免费PDF全文
1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s020,w=11.0S and D020,w=4.94×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H2 evolution, 380 for N2 reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s020,w=4.8S and D020,w=5.55×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H2 evolution, 275 for N2 reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77°K. The u.v. spectra of reduced and O2-inactivated proteins were identical, and inactivation by O2 generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O2 decreased the depth of the trough. 5. The reduction of N2 and acetylene, and H2 evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo–Fe-containing protein; excess of the Mo–Fe-containing protein was inhibitory. All reductions were accompanied by H2 evolution. The combined proteins had no ATP-independent hydrogenase activity. 相似文献
15.
Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. 总被引:4,自引:1,他引:3 下载免费PDF全文
The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents. 相似文献
16.
N2O emission from soil following combined application of fertiliser-N and ground weed residues 总被引:1,自引:0,他引:1
Emissions of N2O and CO2 were measured following combined applications of 15N-labelled fertiliser (100 μg N g−1; 10 atom % excess 15N) and organic olive crop weed residues (Avena sativa, Ononis viscosa, Ridolfia segetum and Olea europea; 100 μg N g−1) to a silt loam soil under controlled environment conditions. The objective was to determine the effect of varying combinations
of inorganic fertiliser and plant residues on these emissions and soil mineral N dynamics. Emissions were generally increased
following application of residues alone, with 23 ng N2O–N g−1 soil (2 ng N2O–N g−1 soil mg−1 biomass) and 389 μg CO2–C g−1 soil (39 μg CO2–C g−1 soil mg−1 biomass) emitted over 28 days after addition of the Ridolfia residues in the absence of fertiliser-N. N2O emissions from these residue-only treatments were strongly negatively correlated with residue lignin content (r = −0.91; P < 0.05), total carbon content (r = −0.90; P < 0.05) and (lignin + polyphenol)-to-N ratio (r = −0.70; P < 0.1). However, changes in the net input of these compounds through application of 25:75, 50:50 and 75:25 proportional mixtures
of Avena and Ononis residues had no effect on emissions compared to their single (0:100 or 100:0) applications. Addition of fertiliser-N increased
emissions (by up to 30 ng N2O–N g−1 28 days−1; 123%), particularly from the low residue-N treatments (Avena and Ridolfia) where a greater quantity of biomass was applied, resulting in emissions above that of the sum from the unfertilised residue
and fertilised control treatments. In contrast, fertiliser application had no impact on emissions from the Olea treatment with the highest polyphenol (2%) and lignin (11%) contents due to strong immobilisation of soil N, and the 15N–N2O data indicated that residue quality had no effect on the denitrification of applied fertiliser-N. Such apparent inconsistencies
mean that before the potential for manipulating N input (organic + inorganic) to lower gaseous N losses can be realised, first
the nature and extent of interactions between the different N sources and any interactions with other compounds released from
the residues need to be better understood. 相似文献
17.
Kumar P Mohammadi M Viger JF Barriault D Gomez-Gil L Eltis LD Bolin JT Sylvestre M 《Journal of molecular biology》2011,405(2):531-44
The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAELB400) are largely unknown. BphAEp4, a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAELB400, yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAELB400 and BphAEp4 and examine the biochemical properties of two BphAELB400 variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity. 相似文献
18.
Joo-Yeon Han Astrid Gräslund Lars Thelander A. G. Sykes 《Journal of biological inorganic chemistry》1997,2(3):287-294
Four reductions of the R2 subunit of mouse ribonucleotide reductase have been studied and found to exhibit different behaviour
from that of Escherichia coli R2. An important difference is that there is no stable met-R2 (Fe2
II I) form of mouse R2. With hydroxyurea, hydrazine and hydroxylamine uniphasic kinetics are observed for the combined reduction
of radical Tyr ˙ and Fe2
II I components to tyrosine and Fe2
II respectively. The rate constants, determined at 370 nm (emphasising FeIII decay) and 417 nm (emphasising Tyr ˙ decay), differ by factors of 2–3, allowing some mechanistic features to be defined. The studies with hydrazine are particularly
important. In the case of E. coli R2, a first phase corresponding to two-equivalent reduction of the met-R2 component has been observed [18]. It is likely
that the four times slower second phase reaction of active E. coli R2 also corresponds to the Fe2
II I → Fe2
II change and is followed by fast intramolecular Fe2
II reduction of the higher potential Tyr ˙. The latter changes are believed to hold also for (active) mouse R2. The FeIIFeIII semi forms have been detected at low levels by EPR for mouse R2 (9%) and E. coli (∼5%) in previous studies. Further substrate reduction of FeIIFeIII occurs at a comparable rate to account for the transient behaviour of FeIIFeIII. For mouse R2 the combined FeIII decay processes (which we are unable to separate) give smaller uniphasic rate constants at 370 nm than at 417 nm. A fitted-base-line
(FBL) treatment of absorbance changes at 417 nm targets more closely the Tyr ˙ decay as a means of monitoring the rate-determining step. The FBL method gives rate constants k (M–1 s–1) at 25 °C and pH 7.5 for hydroxyurea (1.46), hydrazine (0.163) and hydroxylamine (4.4). Surprisingly, phenylhydrazine, with
a less strong reduction potential (0.25 V), gives a substantially faster reduction of the Tyr ˙ as the only redox step (rate constant 27 M–1 s–1). In this case a slower second phase at 370 nm is independent of reductant and corresponds to rate-controlling release of
FeIII. Overall the results indicate a more reactive redox centre for mouse R2 and help develop further an understanding of factors
affecting the reactivity of R2.
Received: 11 October 1996 / Accepted: 11 February 1997 相似文献
19.
Razavi Seyedeh Saleheh Hashemianzadeh Seyed Majid Karimi Hedayat 《Journal of molecular modeling》2011,17(5):1163-1172
Canonical Monte Carlo (CMC) simulations were carried out to investigate the behavior of CO2 and N2 mixtures upon adsorption on single walled carbon nanotubes (CNTs). In the simulation, all the particle–particle interactions
between CO2, N and C were modeled using Lennard-Jones (LJ) potential. To provide deep insight into the effect of pore width, temperature,
pressure and bulk composition on the adsorption behavior of CO2 /N2 mixtures, five different CNTs [(6,6), (7,7), (8,8) (9,9) and (10,10) CNT] with diameters ranging from 0.807 to 1.35 nm, three
temperatures (300 323 and 343 K), six pressures (0.15, 2, 4, 6, 8 and 10 MPa), and three bulk mole compositions of carbon
dioxide (0.3 0.5 and 0.7) were tested. The results from all the simulation conditions investigated in this work show that
CNTs preferentially adsorb carbon dioxide relative to nitrogen in a binary mixture. The results are consistent with the hypothesis
that stronger interaction of one component with the nanotube surface results in a higher adsorption capacity compared to the
other component. An optimized pore size of D = 8.07 nm corresponding to (6, 6) CNT, at T = 300 K and P = 0.15 MPa at a bulk
mole composition of yCO2 =0.3 was identified in which carbon nanotubes demonstrate the greatest selectivity for separation of carbon dioxide relative
to nitrogen. In addition, it is worth pointing out that, under similar simulation conditions, CNTs exhibit higher selectivity
compared to other carbon-based materials [carbon membrane polyimide (PI) and PI/multi-wall carbon nanotubes (MWCNTs)] for
CO2 adsorption. As a prototype, the selectivity of an equimolar mixture of CO2 /N2 for adsorption on (6, 6) CNTs at 300 K and 0.15 MPa reaches 9.68, which is considerably larger than that reported in carbon
membrane. Therefore, it can be concluded that carbon nanotubes can act as a capable adsorbent for adsorption/desorption of
CO2 in comparison with other carbon-based materials in the literature. 相似文献
20.
David Blackburn Matthew Hamilton Chris Harwood Trevor Innes Brad Potts Dean Williams 《Tree Genetics & Genomes》2010,6(5):757-765
A trial was undertaken to assess the extent to which variation in sawn-board quality traits of plantation-grown Eucalyptus nitens is under genetic control and amenable to genetic improvement. Five hundred and sixty trees from 129 families and three central
Victorian races were sampled from an open-pollinated progeny trial in Tasmania, Australia. Acoustic wave velocity (AWV) was
assessed on standing trees and sawlogs. Wedges from disks extracted from sawlogs were assessed for basic density and checking.
Processed boards from 496 of the trees were assessed for board stiffness (static modulus of elasticity, MOE), and internal
and surface checking. Genetic differences among races were significant for AWV and MOE traits. The Southern race had the highest
mean values for these traits. Significant additive genetic variation within races was observed in all traits, demonstrating
that the quality of plantation-grown E. nitens boards could be improved through breeding. Estimated narrow-sense heritabilities were 0.85 for standing-tree AWV, 0.71 for
log AWV, 0.37 for board MOE, and ranged from 0.20 to 0.52 for checking traits. A strongly positive genetic correlation (r
g = 1.05) was observed between standing-tree AWV and board MOE, indicating that AWV could be used as a selection trait to improve
E. nitens board stiffness. The genetic correlation between basic density and board MOE was also positive (r
g = 0.62). However, a significant and adverse genetic correlation (r
g = 0.61) was identified between basic density and surface check length. Wood stiffness and checking traits were more-or-less
genetically independent, and genetic correlations between surface and internal checking were positive but only moderate (r
g = 0.48–0.52). 相似文献