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1.
Protein Sci ; 16(6): 1157-64, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17525464

RESUMEN

The reaction of adenosylcobalamin-dependent dioldehydrase with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X-band EPR spectroscopy was performed in the presence of D(2)O and compared to the spectrum in H(2)O. Results indicate that the unpaired electron of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the (205,203)Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.


Asunto(s)
Cationes Monovalentes/química , Propanodiol Deshidratasa/química , Solventes/química , Sitios de Unión , Cationes Monovalentes/metabolismo , Cobamidas/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Modelos Químicos , Estructura Molecular , Potasio/química , Potasio/metabolismo , Propanodiol Deshidratasa/metabolismo , Protones , Talio/química , Talio/metabolismo
2.
FEBS Lett ; 579(2): 500-6, 2005 Jan 17.
Artículo en Inglés | MEDLINE | ID: mdl-15642366

RESUMEN

Previously, we investigated ubisemiquinone (SQ) EPR spectra associated with NADH-ubiquinone oxidoreductase (complex I) in the tightly coupled bovine heart submitochondrial particles (SMP). Based upon their widely differing spin relaxation rate, we distinguished SQ spectra arising from three distinct SQ species, namely SQ(Nf) (fast), SQ(Ns) (slow), and SQ(Nx) (very slow). The SQ(Nf) signal was observed only in the presence of the proton electrochemical gradient (deltamu(H)(+)), while SQ(Ns) and SQ(Nx) species did not require the presence of deltamu(H+). We have now succeeded in characterizing the redox and EPR properties of SQ species in the isolated bovine heart complex I. The potentiometric redox titration of the g(z,y,x)=2.00 semiquinone signal gave the redox midpoint potential (E(m)) at pH 7.8 for the first electron transfer step [E(m1)(Q/SQ)] of -45 mV and the second step [E(m2)(SQ/QH(2))] of -63 mV. It can also be expressed as [E(m)(Q/QH(2))] of -54 mV for the overall two electron transfer with a stability constant (K(stab)) of the SQ form as 2.0. These characteristics revealed the existence of a thermodynamically stable intermediate redox state, which allows this protein-associated quinone to function as a converter between n=1 and n=2 electron transfer steps. The EPR spectrum of the SQ species in complex I exhibits a Gaussian-type spectrum with the peak-to-peak line width of approximately 6.1 G at the sample temperature of 173 K. This indicates that the SQ species is in an anionic Q(-) state in the physiological pH range. The spin relaxation rate of the SQ species in isolated complex I is much slower than the SQ counterparts in the complex I in situ in SMP. We tentatively assigned slow relaxing anionic SQ species as SQ(Ns), based on the monophasic power saturation profile and several fold increase of its spin relaxation rate in the presence of reduced cluster N2. The current study also suggests that the very slowly relaxing SQ(Nx) species may not be an intrinsic complex I component. The functional role of SQ(Ns) is further discussed in connection with the SQ(Nf) species defined in SMP in situ.


Asunto(s)
Complejo I de Transporte de Electrón/química , Mitocondrias Cardíacas/química , Termodinámica , Ubiquinona/análogos & derivados , Ubiquinona/química , Animales , Bovinos , Coenzimas , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Oxidación-Reducción , Ubiquinona/aislamiento & purificación
3.
J Phys Chem B ; 109(42): 19923-8, 2005 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-16853576

RESUMEN

The core structure of the photosynthetic reaction center is quasisymmetric with two potential pathways (called A and B) for transmembrane electron transfer. Both the pathway and products of light-induced charge separation depend on local electrostatic interactions and the nature of the excited states generated at early times in reaction centers isolated from Rhodobacter sphaeroides. Here transient absorbance measurements were recorded following specific excitation of the Q(y)() transitions of P (the special pair of bacteriochlorophylls), the monomer bacteriochlorophylls (B(A) and B(B)), or the bacteriopheophytins (H(A) and H(B)) as a function of both buffer pH and detergent in a reaction center mutant with the mutations L168 His to Glu and L170 Asn to Asp in the vicinity of P and B(B). At a low pH in any detergent, or at any pH in a nonionic detergent (Triton X-100), the photochemistry of this mutant is faster than, but similar to, wild type (i.e. electron transfer occurs along the A-side, 390 nm excitation is capable of producing short-lived B-side charge separation (B(B)(+)H(B)(-)) but no long-lived B(B)(+)H(B)(-) is observed). Certain buffering conditions result in the stabilization of the B-side charge separated state B(B)(+)H(B)(-), including high pH in the zwitterionic detergent LDAO, even following excitation with low energy photons (800 or 740 nm). The most striking result is that conditions giving rise to stable B-side charge separation result in a lack of A-side charge separation, even when P is directly excited. The mechanism that links B(B)(+)H(B)(-) stabilization to this change in the photochemistry of P in the mutant is not understood, but clearly these two processes are linked and highly sensitive to the local electrostatic environment produced by buffering conditions (pH and detergent).


Asunto(s)
Ambiente , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/genética , Tampones (Química) , Detergentes , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Histidina/genética , Concentración de Iones de Hidrógeno , Mutación , Fotoquímica
4.
Biochemistry ; 43(35): 11206-13, 2004 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-15366930

RESUMEN

A small metal-binding protein (SmbP) with no known similarity to other proteins in current databases was isolated and characterized from the periplasm of Nitrosomonas europaea. The primary structure of this small (9.9 kDa) monomeric protein is characterized by a series of 10 repeats of a seven amino acid motif and an unusually high number of histidine residues. The protein was isolated from N. europaea with Cu(II) bound but was found to be capable of binding multiple equivalents of a variety of divalent and trivalent metals. The protein was overexpressed in Escherichia coli and used for the study of its metal-binding properties by UV/vis, circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy and equilibrium dialysis and isothermal titration calorimetry. The protein was found to bind up to six Cu(II) atoms with dissociation constants of approximately 0.1 microM for the first two metal ions and approximately 10 microM for the next four. Binding of Cu(II) resulted in spectroscopic features illustrating two distinctive geometries, as determined by EPR spectroscopy. The levels of SmbP in the periplasm were found to increase by increasing the levels of copper in the growth media. This protein is proposed to have a role in cellular copper management in the ammonia-oxidizing bacterium N. europaea.


Asunto(s)
Adhesión Bacteriana , Proteínas Bacterianas/química , Cobre/metabolismo , Metaloproteínas/química , Nitrosomonas europaea/química , Proteínas de Unión Periplasmáticas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cationes Bivalentes/metabolismo , Dicroismo Circular , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Metaloproteínas/aislamiento & purificación , Metaloproteínas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Nitrosomonas europaea/metabolismo , Proteínas de Unión Periplasmáticas/aislamiento & purificación , Proteínas de Unión Periplasmáticas/metabolismo , Estructura Secundaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
5.
J Bacteriol ; 186(11): 3408-14, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15150226

RESUMEN

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.


Asunto(s)
Chloroflexus/enzimología , Fotosíntesis , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Superóxido Dismutasa/genética
6.
BMC Plant Biol ; 4: 5, 2004 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-15086960

RESUMEN

BACKGROUND: Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly. RESULTS: Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction. CONCLUSION: CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.


Asunto(s)
Clorofila/biosíntesis , Oxigenasas/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/genética , Radicales Libres/química , Proteínas Fluorescentes Verdes , Immunoblotting , Yoduros/química , Proteínas Hierro-Azufre/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Oxigenasas/química , Oxigenasas/genética , Pisum sativum/citología , Pisum sativum/genética , Pisum sativum/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tilacoides/metabolismo , Transfección
7.
Protein Expr Purif ; 35(1): 131-41, 2004 May.
Artículo en Inglés | MEDLINE | ID: mdl-15039076

RESUMEN

The protein YxaG from Bacillus subtilis, of previously unknown function, was found to have quercetin 2,3-dioxygenase activity when overexpressed in Escherichia coli. The enzyme converts the flavonol quercetin to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide, indicating that it performs the same reaction and yields the same products as the well-characterized copper-containing quercetin 2,3-dioxygenase from Aspergillus. In contrast to the Aspergillus protein, YxaG contains iron, and the enzyme is sensitive to strong Fe(II) chelators, similar to the extensively studied catechol dioxygenases. The active site metal was probed by EPR spectroscopy using the label nitric oxide to confirm the presence of an Fe(II) atom. The kinetic parameters and pH activity profiles are also markedly different from those of the copper-containing quercetin 2,3-dioxygenases from Aspergillus. YxaG represents the first example of a prokaryotic quercetin 2,3-dioxygenase.


Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dioxigenasas , Oxigenasas/aislamiento & purificación , Oxigenasas/metabolismo , Aspergillus/enzimología , Proteínas Bacterianas/genética , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Inhibidores Enzimáticos/metabolismo , Hierro/metabolismo , Estructura Molecular , Oxigenasas/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Quercetina/metabolismo
8.
Inorg Chem ; 43(2): 506-14, 2004 Jan 26.
Artículo en Inglés | MEDLINE | ID: mdl-14731012

RESUMEN

The reaction between Mn(6)L(12) and Mg(6)L(12) (L = N,N-diethylcarbamate) results in isolation of heteronuclear complexes Mn(n)Mg(6)(-)(n)L(12). A series was prepared with different doping factors n by varying the Mn/Mg ratio in the crystallization solutions. Single-crystal X-ray diffraction shows that MnMg(5)L(12) is isostructural with Mn(6)L(12) and Mg(6)L(12). Magnetic susceptibility data on the series Mn(n)Mg(6)(-)(n)L(12) (n = 1-6) are consistent with antiferromagnetic Mn.Mn interactions. At low n, the magnetic data demonstrate the formation of magnetically isolated Mn(2+) centers. This was confirmed by measurement of the EPR spectrum at a doping factor n = 0.06 in solution, as a powder, and as single crystals. These show hyperfine interactions consistent with isolated Mn(2+). The EPR spectrum of Mn(0.06)Mg(5.94)L(12) exhibits a dominant signal at g(eff) = 4, and a wide series of less intense signals spanning 200-6000 G in the X-band regime. This unusual behavior in a weak-field Mn(2+) complex is attributed to the substantial distortions from cubic ligand field geometry in this system. The g(eff) = 4 signals are attributed to a C(2)-symmetric hexacoordinate Mn(2+) ion with D > 0.3 cm(-)(1) and E/D = 0.33. The wide series is assigned to an axial C(4)(v) pentacoordinate Mn(2+) site with D = 0.05 cm(-)(1). Comparison of the g(eff) = 4 signals to the g = 4.1 signals exhibited by the tetramanganese complex in photosystem II belies the fact that they almost certainly arise from different spin systems. In addition, the similarity of the spectrum of Mn(n)Mg(6)(-)(n)L(12) to mononuclear Mn(4+) complexes suggests that considerable care must be exercised in the use of EPR as a fingerprint for the manganese oxidation state, particularly in manganese proteins where molecular composition may not be precisely established.

9.
Biochemistry ; 41(14): 4649-54, 2002 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-11926827

RESUMEN

Human cystathionine beta-synthase is a hemeprotein that catalyzes a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine into cystathionine. Biophysical characterization of this enzyme has led to the assignment of the heme ligands as histidine and cysteinate, respectively, which has recently been confirmed by crystal structure determination of the catalytic core of the protein. Using site-directed mutagenesis, we confirm that C52 and H65 represent the thiolate and histidine ligands to the heme. Conversion of C52 to alanine or serine results in spectral properties of the resulting hemeprotein that are consistent with the loss of a thiolate ligand. Thus, the Soret peak blue-shifts from 428 to 415 and 417 nm in the ferric forms of the C52S and C52A mutants, respectively, and from 450 to 423 nm in the ferrous states of both mutants. Addition of CO to the dithionite-reduced ferrous C52 mutants results in spectra with Soret peaks at 420 nm. EPR spectroscopy of the ferric C52 variants reveals the predominance of a high-spin species. The H65R mutant, a variant described in a homocystinuric patient, has Soret peaks at 424, 421, and 420 nm in the ferric, ferrous, and ferrous CO states, respectively. EPR spectroscopy reveals predominance of the low-spin species. Both C52A and C52S mutations lead to protein with substoichiometric heme (19% with respect to wild type); however, the PLP content is comparable to that of wild-type enzyme. The heme and PLP contents of the H65R mutant are 40% and 75% that of wild-type enzyme. These results indicate that heme saturation does not dictate PLP saturation in these mutant enzymes. Both H65 and C52 variants display low catalytic activity, revealing that changes in the heme binding domain modulate activity, consistent with a regulatory role for this cofactor.


Asunto(s)
Cistationina betasintasa/genética , Variación Genética , Hemo/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Cistationina betasintasa/metabolismo , Humanos , Cinética , Ligandos , Modelos Moleculares , Conformación Proteica , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrofotometría
10.
Inorg Chem ; 36(21): 4866-4874, 1997 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-11670167

RESUMEN

The complexes [VO(H(2)O)ada] (1), [VO(H(2)O)Hheida] (2), and [VO(H(2)O)aeida] (3) (H(2)ada, N-(carbamoylmethyl)iminodiacetic acid; H(3)heida, N-(2-hydroxyethyl)iminodiacetic acid; H(2)aeida, N-(2-aminoethyl)iminodiacetic acid) were synthesized and crystallographically characterized. Crystallographic parameters for 1.2H(2)O: monoclinic, space group P2(1)/c (No. 14), a = 7.327(2) Å, b = 23.386(7) Å, c = 7.258(3) Å, alpha = 90 degrees, beta = 110.95(2) degrees, gamma = 90 degrees, V = 1204.6(7) Å(3), Z = 4, R1 = 0.0353, and wR(2)() = 0.0848. Crystallographic parameters for 2.H(2)O: orthorhombic, space group Pbca (No. 61), a = 10.512(2) Å, b = 11.727(2) Å, c = 16.719(5) Å, alpha = 90 degrees, beta = 90 degrees, gamma = 90 degrees, V = 2060.6(8) Å(3), Z = 8, R1 = 0.0297, and wR(2)() = 0.0758. Crystallographic parameters for 3: monoclinic, space group P2(1)/c (No. 14), a = 6.785(1) Å, b = 9.714(2) Å, c = 14.959(2) Å, alpha = 90 degrees, beta = 95.12(1) degrees, gamma = 90 degrees, V = 982.2(3) Å(3), Z = 4, R1 = 0.0298, and wR(2)() = 0.0762. In each structure, the tetradentate ligand is disposed so that the tertiary nitrogen is bound trans to the vanadyl oxo, and the rest of the donors occupy equatorial coordination positions. In solution, the structural integrity of these compounds is maintained as observed by UV/visible and EPR spectroscopies, and axial ligation by nitrogen is inferred on the basis of ESEEM spectroscopy. The implications of this study with respect to understanding the coordination environment of VO(2+) in the reduced, inactive form of vanadium bromoperoxidase (VBrPO) are discussed, and it is proposed that significant changes in the coordination environment of vanadium in VBrPO occur upon its reduction, which may provide a plausible explanation for its irreversible inactivation.

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