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1.
《BBA》2020,1861(9):148237
Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a3 (Fea3) and copper (CuB). Here, the influence of the ligation at the oxidized Fea33+–CuB2+ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (Em(a)) for the cyanide (the low-spin Fea33+) and the formate-ligated CcO (the high-spin Fea33+). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of Em(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fea3+ between His–Fea3+–His and His–Fea3+–OH− occurs only at pH above 9.5 with the estimated pK >11.0. 相似文献
2.
Summary. The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable
of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome
b
561 proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b
561 located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves
of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction,
containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials
of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing
the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at
about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two
bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully
ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b
561 proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis
genome (24–31 kDa).
Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003
RID="*"
ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary. 相似文献
3.
Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C5H11) by H2O2 to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of
Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25 °C. The rate of oxidation of ferrocenes with longer alkyl radicals is
too slow to be measured. The reaction obeying the [RFc]:[H2O2] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed
second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl
substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects
are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with
organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic
second-order rate constants k
w are (1.9 ± 0.5)×105, (2.7 ± 0.1)×104, and (5.9 ± 0.6)×103 M–1 s–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple
in terms of the Marcus formalism.
Received: 15 July 1996 / Accepted: 15 November 1996 相似文献
4.
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
5.
Jose Neptuno Rodriguez-Lopez Andrew T. Smith R. N. F. Thorneley 《Journal of biological inorganic chemistry》1996,1(2):136-142
Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem
cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these
residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased
the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k
1 = 1.4×102 M–1s–1) compared with both native-glycosylated and recombinant forms of HRPC (k
1 = 1.7×107 M–1s–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to
be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed
pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k
1 = 1.1×104 M–1s–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k
2 = 142 s–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion
of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly,
stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38
explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases.
Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are
involved in the modulation of substrate affinity.
Received: 21 July 1995 / Accepted: 27 November 1995 相似文献
6.
7.
Kabir ME Krishnaswamy S Miyamoto M Furuichi Y Komiyama T 《Applied microbiology and biotechnology》2011,90(2):553-564
Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional
antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific
altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated
against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional
antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative
small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library
through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in
bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data
showed that the binding affinity for this single-domain antibody was 272-fold higher (K
d = 1.07 × 10−10 M) and antifungal activity was three- to fivefold more efficient (IC50 = 0.46 × 10−6 to 1.17 × 10−6 M) than that for the conventional antibody (K
d = 2.91 × 10−8 M and IC50 = 2.14 × 10−6 to 3.78 × 10−6 M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development
of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain
synthetic antibodies will find their way into a number of biotechnological or medical applications. 相似文献
8.
By means of microcalorimetry, the effect of four copper(II) complexes on Tetrahymena growth was investigated. The extent and duration of the inhibitory effect on the metabolism, judged by the rate constant,
k, and the half inhibition concentration, IC50, varied with the different complexes. The results showed that the half inhibition concentrations IC50 of CuCl2, (C9H6NO)2Cu and [Cu(phen)2]Cl2⋅6H2O were 9.9 × 10−4, 2.0 × 10−4, and 2.6 × 10−4 mol/L, respectively. The sequence of antibiotic activity of these three complexes was: (C9H6NO)2Cu > [Cu(phen)2]Cl2⋅6H2O > CuCl2. The growth rate constants of [Cu(phen)3]Cl2⋅6H2O did not change obviously with the increase of concentrations, but [Cu(phen)3]Cl2⋅6H2O also can prolong the time of Tetrahymena growth. 相似文献
9.
Tiecheng Qiao Robert Witkowski Robin Henderson G. McLendon 《Journal of biological inorganic chemistry》1996,1(5):432-438
The kinetics of methemoglobin reduction by cytochrome b
5 has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k
f = 2.44×104 M–1 s–1 and a reverse rate constant k
b = 540 M–1s–1 have been observed at 10 mm, pH 6.20, 25 °C. The ratio k
f/k
b = k
eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b
5 and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b
5 to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such
collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin.
Received: 20 February 1996 / Accepted: 4 June 1996 相似文献
10.
Miriam Altstein Tal Gabay Orna Ben-Aziz Shai Daniel Irina Zeltser Chaim Gilon 《Invertebrate neuroscience : IN》1999,4(1):0033-0040
The binding of [3H]tyrosyl-PBAN28-33NH2 to pheromone gland membranes of the moth Heliothis peltigera was investigated. The study describes the development of a pheromone biosynthesis-activating neuropeptide (PBAN) radioreceptor
assay and demonstrates the presence of a putative PBAN binding site on the pheromone gland. It also describes synthesis of
a radioligand and optimization of binding conditions with respect to membrane preparation, number of gland equivalents, kinetics
of ligand binding and composition of the binding solution. Binding was found to be optimal when membranes were freshly prepared
from frozen glands, incubated at a concentration of one gland equivalent per reaction tube in the presence of 10 mM HCO3
− ions. Equilibrium of ligand binding was obtained after 20 min. Presence of other components such as NaCl, KCl or SH reagents
did not have any effect on binding. Binding was found to be saturable, with a Kd of 5.73 ± 1.05 × 10−6 M and a Bmax of 1.85 ± 0.22 nmol/mg protein. Binding was effectively displaced by unlabeled PBAN1-33NH2 and PBAN28-33ΝΗ2 with a Ki of 4.3 ± 1.1 × 10−6 M and 4.9 ± 2.6 × 10−6 M, respectively.
Accepted: 4 February 1999 相似文献
11.
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
12.
This mini-review briefly summarizes our current knowledge on the reaction pattern of light-driven water splitting and the
structure of Photosystem II that acts as a water:plastoquinone oxidoreductase. The overall process comprises three types of
reaction sequences: (a) light-induced charge separation leading to formation of the radical ion pair P680+•QA−•; (b) reduction of plastoquinone to plastoquinol at the QB site via a two-step reaction sequence with QA−• as reductant and (c) oxidative water splitting into O2 and four protons at a manganese-containing catalytic site via a four-step sequence driven by P680+• as oxidant and a redox active tyrosine YZ acting as mediator. Based on recent progress in X-ray diffraction crystallographic structure analysis the array of the cofactors
within the protein matrix is discussed in relation to the functional pattern. Special emphasis is paid on the structure of
the catalytic sites of PQH2 formation (QB-site) and oxidative water splitting (Mn4O
x
Ca cluster). The energetics and kinetics of the reactions taking place at these sites are presented only in a very concise
manner with reference to recent up-to-date reviews. It is illustrated that several questions on the mechanism of oxidative
water splitting and the structure of the catalytic sites are far from being satisfactorily answered. 相似文献
13.
An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide
gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of 1H-NMR spectra in D2O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k
cat/K
m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus.
Received: 19 June 1998 / Received revision: 15 September 1998 / Accepted: 17 September 1998 相似文献
14.
In this study, sludge was taken from a municipal wastewater treatment plant that contained a nearly equal number of archaeal
amoA genes (5.70 × 106 ± 3.30 × 105 copies mg sludge−1) to bacterial amoA genes (8.60 × 106 ± 7.64 × 105 copies mg sludge−1) and enriched in three continuous-flow reactors receiving an inorganic medium containing different ammonium concentrations:
2, 10, and 30 mM NH4+–N (28, 140, and 420 mg N l−1). The abundance and communities of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in enriched nitrifying
activated sludge (NAS) were monitored at days 60 and 360 of the operation. Early on, between day 0 and day 60 of reactor operation,
comparative abundance of AOA amoA genes to AOB amoA genes varied among the reactors depending on the ammonium levels found in the reactors. As compared to the seed sludge, the
number of AOA amoA genes was unchanged in the reactor with lower ammonium level (0.06 ± 0.04 mgN l−1), while in the reactors with higher ammonium levels (0.51 ± 0.33 and 0.25 ± 0.10 mgN l−1), the numbers of AOA amoA genes were deteriorated. By day 360, AOA disappeared from the ammonia-oxidizing consortiums in all reactors. The majority
of the AOA sequences from all NASs at each sampling period fell into a single AOA cluster, however, suggesting that the ammonium
did not affect the AOA communities under this operational condition. This result is contradictory to the case of AOB, where
the communities varied significantly among the NASs. AOB with a high affinity for ammonia were present in the reactors with
lower ammonium levels, whereas AOB with a low affinity to ammonia existed in the reactors with higher ammonium levels. 相似文献
15.
Smirnova IA Zaslavsky D Fee JA Gennis RB Brzezinski P 《Journal of bioenergetics and biomembranes》2008,40(4):281-287
The ba
3-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa
3–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O2 to water is catalysed at a haem a
3-CuB catalytic site. The three-dimensional structure of the ba
3 oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial,
Rhodobacter sphaeroides and Paracoccus denitrificans aa
3 oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba
3
-cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence
of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 104 s−1) and formation of the peroxy (PR) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 104 s−1, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s−1, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle
was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the PR and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba
3 oxidases, the proton-uptake reactions occur over the same time scales as in the aa
3-type oxidases.
Smirnova and Zaslavsky contributed equally to the work described in this paper. 相似文献
16.
trans -[PtCl4(NH3)(thiazole)] (1), trans-[PtCl4(cha)(NH3)] (2), cis-[PtCl4(cha)(NH3)] (3) (cha =cyclohexylamine), and cis-[PtCl4(NH3)2] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes
and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of
these protolytic equilibria. The thiolate species GS− is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS− are (1.43±0.01)×107, (3.86±0.03)×106, (1.83±0.01)×106, and (1.18±0.01)×106 M−1 s−1, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination
reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results
together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity
profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction
within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter
to increase anticancer activity, at least for this class of compounds.
Received: 8 December 1999 / Accepted: 15 February 2000 相似文献
17.
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
18.
Jingyuan Ai John A. Broadwater Thomas M. Loehr Joann Sanders-Loehr B. G. Fox 《Journal of biological inorganic chemistry》1997,2(1):37-45
The stearoyl-acyl carrier protein Δ9 desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O2–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities
in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear
to share common features of their reaction cycles, including the binding of O2 to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate
the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron
center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results
in the appearance of new absorption bands at 325 nm and 440 nm (k
app≈3.5 s–1 in 0.7 M NaN3, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η1–terminal structure at pH 7.8 (14N3
– asymmetric stretch at 2073 cm–1, resolved into two bands with 15N14N2
–) and a μ-1,3 bridging structure at pH<7 (14N3
– asymmetric stretch at 2100 cm–1, shifted as a single band with 15N14N2
–). Both adducts also exhibit an Fe–N3 stretching mode at ≈380 cm–1, but no accompanying Fe–O–Fe stretching mode, presumably due to either protonation or loss of the oxo bridge. The ability
to form a μ-1,3 bridging azide supports the likelihood of a μ-1,2 bridging peroxide as a catalytic intermediate in the Δ9D
reaction cycle and underscores the adaptability of binuclear sites to different bridging geometries.
Received: 23 August 1996 / Accepted: 4 October 1996 相似文献
19.
ETA subtype selective antagonists constitute a novel and potentially important class of agents for the treatment of pulmonary
hypertension, heart failure, and other pathological conditions. In this paper, 60 benzodiazepine derivatives displaying potent
activities against ETA and ETB subtypes of endothelin receptor were selected to establish the 3D-QSAR models using CoMFA and CoMSIA approaches. These models
show excellent internal predictability and consistency, external validation using test-set 19 compounds yields a good predictive
power for antagonistic potency. Statistical parameters of models were obtained with CoMFA-ETA (q
2 = 0.787, r
2 = 0.935, r
2
pred
= 0.901), CoMFA-ETB (q
2 = 0.842, r
2 = 0.984, r
2
pred
= 0.941), CoMSIA-ETA (q
2 = 0.762, r
2 = 0.971, r
2
pred
= 0.958) and CoMSIA-ETB (q
2 = 0.771, r
2 = 0.974, r
2
pred
= 0.953) respectively. Field contour maps (CoMFA and CoMSIA) corresponding to the ETA and ETB subtypes reflects the characteristic similarities and differences between these types. The results of this paper provide
valuable information to facilitate structural modifications of the title compounds to increase the inhibitory potency and
subtype selectivity of endothelin receptor. 相似文献
20.
Juozas Kulys Kastis Krikstopaitis Arturas Ziemys 《Journal of biological inorganic chemistry》2000,5(3):333-340
N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k
cat/K
m) varied from 1 ×107 M−1 s−1to 2.6×108 M−1 s−1 at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable
groups with pK
a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed
that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were
consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent
bimolecular rate constants varied from 1.8×105 M−1 s−1 to 2.0×107 M−1 s−1 at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent
bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed
in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO,
fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×108 M−1 s−1 and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×107 M−1 s−1 and 0.29 eV.
Received: 20 September 1999 / Accepted: 24 February 2000 相似文献