RESUMEN
The recently discovered prokaryotic signal transducer HemAT, which has been described in both Archaea and Bacteria, mediates aerotactic responses. The N-terminal regions of HemAT from the archaeon Halobacterium salinarum (HemAT-Hs) and from the Gram-positive bacterium Bacillus subtilis (HemAT-Bs) contain a myoglobin-like motif, display characteristic heme-protein absorption spectra, and bind oxygen reversibly. Recombinant HemAT-Hs and HemAT-Bs shorter than 195 and 176 residues, respectively, do not bind heme effectively. Sequence homology comparisons and three-dimensional modeling predict that His-123 is the proximal heme-binding residue in HemAT from both species. The work described here used site-specific mutagenesis and spectroscopy to confirm this prediction, thereby providing direct evidence for a functional domain of prokaryotic signal transducers that bind heme in a globin fold. We postulate that this domain is part of a globin-coupled sensor (GCS) motif that exists as a two-domain transducer having no similarity to the PER-ARNT-SIM (PAS)-domain superfamily transducers. Using the GCS motif, we have identified several two-domain sensors in a variety of prokaryotes. We have cloned, expressed, and purified two potential globin-coupled sensors and performed spectral analysis on them. Both bind heme and show myoglobin-like spectra. This observation suggests that the general function of GCS-type transducers is to bind diatomic oxygen and perhaps other gaseous ligands, and to transmit a conformational signal through a linked signaling domain.
Asunto(s)
Archaea/metabolismo , Bacterias/metabolismo , Técnicas Biosensibles , Globinas/metabolismo , Hemo/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al Hemo , Hemoproteínas/química , Hemoproteínas/genética , Hemoproteínas/aislamiento & purificación , Hemoproteínas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The focus of this study is to examine volume and enthalpy profiles of ligand binding associated with CO-Fe(II) tetrakis-(4-sulfonato phenyl)-porphyrin (COFe(II)4SP) in aqueous solution. Temperature dependent photothermal beam deflection was employed to probe the overall enthalpy and volume changes associated with CO-photolysis and recombination. The analysis demonstrates that ligand recombination occurs with a pseudo first order rate constant of (2.5+/-0.2)x10(4) s(-1) (at 25 degrees C) with a corresponding volume decrease of 6+/-1 ml/mol. The activation enthalpy (DeltaH(double dagger)) and volume (DeltaV(double dagger)) change for CO recombination (determined from temperature/pressure dependent transient absorption spectroscopy) are found to be 3.9 kcal/mol and 8.2 ml/mol, respectively. These data are consistent with a mechanism in which photolysis yields a five-coordinate high spin (H(2)O)Fe(II)4SP complex that recombines in a single step to form the low spin (CO)(H(2)O)Fe(II)4SP complex. Base elimination, often associated with CO photolysis from hemes, is not observed in this system. The overall volume changes suggest a transition state with significant high spin character. Furthermore, these results demonstrate the utility of coupling photothermal techniques with variable pressure/temperature transient absorption spectroscopy to probe heme reaction dynamics.
Asunto(s)
Monóxido de Carbono/metabolismo , Metaloporfirinas/química , Monóxido de Carbono/química , Hierro/metabolismo , Cinética , Ligandos , Metaloporfirinas/metabolismo , Fotoquímica , Presión , Análisis Espectral , Temperatura , Termodinámica , Agua/química , Agua/metabolismoRESUMEN
Haem-containing proteins such as haemoglobin and myoglobin play an essential role in oxygen transport and storage. Comparison of the amino-acid sequences of globins from Bacteria and Eukarya suggests that they share an early common ancestor, even though the proteins perform different functions in these two kingdoms. Until now, no members of the globin family have been found in the third kingdom, Archaea. Recent studies of biological signalling in the Bacteria and Eukarya have revealed a new class of haem-containing proteins that serve as sensors. Until now, no haem-based sensor has been described in the Archaea. Here we report the first myoglobin-like, haem-containing protein in the Archaea, and the first haem-based aerotactic transducer in the Bacteria (termed HemAT-Hs for the archaeon Halobacterium salinarum, and HemAT-Bs for Bacillus subtilis). These proteins exhibit spectral properties similar to those of myoglobin and trigger aerotactic responses.
Asunto(s)
Proteínas Arqueales/aislamiento & purificación , Bacillus subtilis/química , Proteínas Bacterianas/aislamiento & purificación , Halobacterium salinarum/química , Hemoproteínas/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/genética , Bacillus subtilis/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Quimiotaxis , Escherichia coli/fisiología , Halobacterium salinarum/fisiología , Proteínas de Unión al Hemo , Hemoproteínas/química , Hemoproteínas/genética , Proteínas Quimiotácticas Aceptoras de Metilo , Datos de Secuencia Molecular , Oxígeno/metabolismo , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
The present work examines the activation volumes associated with intramolecular electron transfer (ET) within the CO-mixed-valence form of bovine heart cytochrome c oxidase (CcO). Activation volumes for intramolecular ET between cytochrome a(3) and cytochrome a (k=(6. 7+/-0.9)x10(5) s(-1) at ambient pressure) and between cytochrome a and Cu(A) (k=(5.9+/-1.7)x10(4) s(-1)) are found to be +41+/-5 ml/mol and +28+/-4 ml/mol, respectively. Examination of the crystal structures of both the fully oxidized and fully reduced forms of bovine heart CcO suggest that the activation volume for the ET between cytochrome a(3) and cytochrome a arises from structural changes localized at cytochrome a(3) upon heme reduction. Similarly, the activation volume for the ET between cytochrome a and Cu(A) is primarily due to structural changes localized at Cu(A) upon reduction of this site. Reduction/oxidation of cytochrome a does not appear to make any significant contribution to the activation volume. Overall, these results suggest conformational regulation of ET by both Cu(A) and cytochrome a(3) but not cytochrome a.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Miocardio/enzimología , Animales , Bovinos , Transporte de Electrón , Complejo IV de Transporte de Electrones/química , Activación Enzimática , Oxidación-Reducción , Conformación ProteicaRESUMEN
In the present study, structural aspects of the two soluble transducers, HtrX and HtrXI, from the archaeon H. salinarum have been examined using UV circular dichroism and steady-state fluorescence spectroscopies. Circular dichroism (CD) data indicate that both HtrX and HtrXI exhibit salt-dependent protein folding. Under low-ionic-strength conditions (0.2 M NaCl or KCl) the CD spectra of HtrXI is similar to that of the Gdn-HCl- or urea-denatured forms and is indicative of random coil structure. In contrast, the CD spectrum of HtrX under low-ionic-strength conditions contains roughly 85% alpha-helical character, indicating a significant degree of folding. Addition of NaCl or KCl to solutions of HtrX or HtrXI results in CD features consistent with predominately alpha-helical character (>95%) for both proteins. In addition, the transition points (i.e., ionic strengths at which the protein converts from random coil to alpha-helical character) are quite distinct and dependent upon the type of salt present (i.e., either NaCl or KCl). Accessibility of tryptophan residues to the solvent was also examined for both HtrX and HtrXI in both folded and unfolded states using KI quenching. The Stem-Volmer constants obtained suggest that the tryptophans (Trp35 in HtrX and both Trp47 and Trp74 in HtrXI) are partially exposed to the solvent, indicating that they are located near the surface of the protein in all three cases. Furthermore, fluorescence quenching with the single Trp mutants Trp74AIa and Trp47AIa of HtrXI indicates different environments for these two residues.
Asunto(s)
Proteínas Arqueales/química , Halobacterium salinarum/química , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Proteínas de la Membrana/química , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Transducción de Señal , Cloruro de Sodio/metabolismo , Cloruro de Sodio/farmacología , Volumetría , Triptófano/químicaRESUMEN
Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins. Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.
Asunto(s)
Proteínas Arqueales/química , Factores Quimiotácticos/química , Complejo IV de Transporte de Electrones/química , Oxígeno/farmacología , Transducción de Señal , Secuencia de Aminoácidos , Proteínas Arqueales/fisiología , Clonación Molecular , Remoción de Radical Alquila , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
Ruthenation of exterior amino acid residues of heme proteins provides an effective means by which biological ET reactions can be studied within the context of highly complex protein environments. Resonance Raman spectroscopy can probe both ET kinetics and structural dynamics at the molecular level. Here we present the first comprehensive use of time-resolved and transient resonance Raman spectroscopies to examine photoinduced ET in cytochromes. Two ruthenated cytochromes c, Ru(lys72)-cyt.c and Ru(cyt102)cyt.c, were studied with TRRS using 10 ns laser pulses and with TRRRS on a 10 ns to 10 ms time scale. It was found that resonance Raman protocols can effectively trace ET kinetics and associated heme--protein structural dynamics. Care must be exercised, however, when drawing comparisons to measurements made by other methods (i.e., transient absorbance). The TRRS studies directly probe the heme and its local environment and reveal that the heme dynamics accompanying ET are very rapid relative to phenomenological ET kinetics. The heme and its local environment evolve to their equilibrium (ferrous) structure in less than 10 ns subsequent to ET, with no evidence for the existence of metastable heme pocket geometries analogous to those observed in the dynamic response of hemoglobins and oxidases. Finally, species-specific differences are observed in the photoinduced ET kinetics and heme structural dynamics. However, these differences are confined to nanosecond or faster time scales.
Asunto(s)
Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Rutenio/farmacología , Transporte de Electrón , Cinética , Luz , Lisina , Modelos Químicos , Saccharomyces cerevisiae/metabolismo , Espectrometría Raman , Factores de TiempoRESUMEN
Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.
Asunto(s)
Cafeína/química , Cafeína/farmacología , ADN/efectos de los fármacos , Sustancias Intercalantes/química , Sustancias Intercalantes/farmacología , Naranja de Acridina/química , Naranja de Acridina/farmacología , Fenómenos Biofísicos , Biofisica , ADN/química , Etidio/química , Etidio/farmacología , Colorantes Fluorescentes/química , Técnicas In Vitro , Modelos Moleculares , Mutágenos/química , Mutágenos/farmacología , Espectrometría de Fluorescencia , Espectrofotometría , TermodinámicaRESUMEN
Transient resonance Raman, Raman difference, circular dichroism (CD), and optical absorption studies have been carried out on the electrostatic complexes formed by yeast cytochrome c peroxidase (CCP) with horse cytochrome c (Cytc) in low ionic strength solutions. In all the complexes examined [e.g., CCP(II)/Cytc(II), CCP(III)/Cytc(II), CCP(III)/Cytc(III)], the local heme environments of both proteins are largely unperturbed upon complexation. Specifically, CCP preserves a completely pentacoordinate high-spin heme in both its ferric and ferrous forms in CCP/Cytc complexes and uncomplexed mixtures. We found no evidence corroborating the previously reported increase in the low-spin fraction of CCP heme upon complexation with Cytc [Hildebrandt et al. (1992) Biochemistry 31, 2384-2392]. Instead, our Raman data strongly suggest that the H-bonding networks in the distal and proximal pockets of CCP are well maintained in the complexes. On the other hand, CD spectra of CCP(III)/Cytc(III) complexes showed substantial variations (relative to the uncomplexed mixtures) in the far-UV region, reflecting some protein conformational rearrangements. In addition, the spectral data suggest that complexation with Cytc affects the previously observed pH-dependent flexibility of the heme structure of CCP and thus influences the photodynamics of the CCP active site.
Asunto(s)
Grupo Citocromo c/química , Citocromo-c Peroxidasa/química , Hemo/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dicroismo Circular , Grupo Citocromo c/genética , Citocromo-c Peroxidasa/genética , Electroquímica , Caballos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Datos de Secuencia Molecular , Estructura Molecular , Concentración Osmolar , Fotoquímica , Conformación Proteica , Saccharomyces cerevisiae/enzimología , Soluciones , Espectrofotometría , Espectrometría Raman , TermodinámicaRESUMEN
It is now widely believed that the first two electrons transferred to the dioxygen reduction site in cytochrome c oxidase (CcO) are not coupled to proton translocation. The activation of the pump cycle correlates with the binding of dioxygen to the binuclear center. In order to investigate conformational changes in CcO associated with the formation of dioxygen intermediates during the catalytic cycle of CcO, the effects of hydrogen peroxide binding to CcO have been examined using UV optical absorption and second derivative techniques. Our data indicates that in the presence low concentrations of H2O2 (2:1 molar ratio) an initial CcO-peroxide species is formed in which the 280-nm absorption band is red shifted. This red shift occurs prior to spectral changes associated with H2O2 binding to cytochrome a3. Upon addition of higher concentrations of H2O2 (> 10 equivalents of H2O2 per equivalent of CcO) oxidized CcO is converted to F-state enzyme with no corresponding shift at 280 nm. It is suggested that H2O2 initially binds to CuB2+ resulting in a conformational change in the enzyme giving rise to a red-shifted 280 nm band. The absence of any conformational changes in F-state enzyme is consistent with the lack of bridging interactions with CuB2+ in this intermediate.
Asunto(s)
Complejo IV de Transporte de Electrones/química , Peróxido de Hidrógeno/farmacología , Conformación Proteica , Animales , Bovinos , Complejo IV de Transporte de Electrones/efectos de los fármacos , Complejo IV de Transporte de Electrones/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidación-Reducción , Unión Proteica , Conformación Proteica/efectos de los fármacos , Espectrofotometría UltravioletaRESUMEN
It has been known for some time that dicyclohexylcarbodiimide (DCCD) inhibits the proton translocation function of the cytochrome c oxidase complex (CcO) and that there is one major site in subunit III which is modified upon reaction with DCCD (Glu-90 for the bovine enzyme). We have examined the reaction of bovine CcO with N-cyclohexyl-N'-(4-dimethylamino-alpha-napthyl)carbodiimide (NCD-4), a fluorescent analog of DCCD. NCD-4 labeling of CcO is strongly inhibited by DCCD implicating Glu-90 of subunit III as the site of chemical modification by NCD-4. The fluorescence of reconstituted NCD-4-labeled bovine CcO is strongly quenched by hydrophobic nitroxides, whereas hydrophilic nitroxides and iodide ions have a reduced quenching ability. It is concluded that the Glu-90 of subunit III resides near the protein-lipid interface of the membrane spanning region of the enzyme. Different quenching abilities of 5-, 7-, 10-, 12-, and 16-4,4-dimethyl-3-oxazolinyloxy-stearic acids suggest that the NCD-4 label is located in the membrane bilayer in the region near the middle of the hydrocarbon tail of stearic acid. In light of these results, it is unlikely that Glu-90 is part of a proton channel that is associated with the proton pumping machinery of the enzyme but the outcome of this study does not eliminate an allosteric regulatory role for this residue.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Espectrometría de Fluorescencia/métodos , Secuencia de Aminoácidos , Animales , Carbodiimidas , Bovinos , Óxidos N-Cíclicos , Diciclohexilcarbodiimida/farmacología , Espectroscopía de Resonancia por Spin del Electrón/métodos , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/química , Colorantes Fluorescentes , Cinética , Sustancias Macromoleculares , Matemática , Mitocondrias Cardíacas/enzimología , Modelos Teóricos , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Espectrofotometría/métodos , Marcadores de SpinRESUMEN
The focus of this study was to examine the functional role of the unusual peripheral substitution of heme A. The effects of heme A stereochemistry on the reconstitution of the porphyrin have been examined in the heme A-apo-myoglobin complex using optical absorption and resonance Raman and electron paramagnetic resonance spectroscopies. The addition of one equivalent of heme A to apo-Mb produces a complex which displays spectroscopic signals consistent with a distribution of high- and low-spin heme chromophores. These results indicate that the incorporation of heme A into apo-Mb significantly perturbs the protein refolding.
Asunto(s)
Hemo/análogos & derivados , Mioglobina/química , Análisis Espectral , Animales , Bovinos , Dimetilsulfóxido , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Férricos/química , Compuestos Ferrosos/química , Hemo/química , Conformación Proteica , Espectrometría Raman , Relación Estructura-ActividadRESUMEN
Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Raman bands indicative of low-spin, six-coordinate iron before Cu2+ binding exhibit monotonic intensity shifts to bands representing high-spin, five-coordinate iron. The latter coordination is in contrast to that found in hemoglobin and myoglobin, and explains the Cu(2+)-induced decrease and broadening of the Fe(3+)-mesoporphyrin.HRG Soret band concomitant with the increase in the high-spin marker band at 620 nm. After dithionite reduction, the Fe(2+)-mesoporphyrin.HRG complex displays high frequency resonance Raman bands characteristic of low-spin heme and no iron-histidine stretch, which together suggest six-coordinate iron. Furthermore, the local heme environment of the complex is not altered by the binding of Cu1+. CO-bound Fe(2+)-mesoporphyrin.HRG exhibits bands in the high and low frequency regions similar to those of other CO-bound heme proteins except that the iron-CO stretch at 505 cm-1 is unusually broad with delta nu approximately 30 cm-1. The dynamics of CO photolysis and rebinding to Fe(2+)-mesoporphyrin.HRG are also distinctive. The net quantum yield for photolysis at 10 ns is low relative to most heme proteins, which may be attributed to very rapid geminate recombination. A similar low net quantum yield and broad iron-CO stretch have so far only been observed in a dimeric cytochrome c' from Chromatium vinosum. Furthermore, the photolytic transient of Fe(2+)-mesoporphyrin.HRG lacks bands corresponding to high-spin, five-coordinate iron as is found in hemoglobin and myoglobin under similar experimental conditions, suggesting iron hexacoordination before CO recombination. These data are consistent with a closely packed distal heme pocket that hinders ligand diffusion into the surrounding solvent.
Asunto(s)
Cobre/química , Proteínas/química , Animales , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Proteínas Sanguíneas/química , Monóxido de Carbono/química , Glicoproteínas/química , Hemo/química , Técnicas In Vitro , Hierro/química , Mesoporfirinas/química , Oxidación-Reducción , Fotólisis , Conejos , Espectrometría RamanRESUMEN
This paper explores the proton pumping function of cytochrome c oxidase [ferrocytochrome-c:oxygen oxidoreductase (EC 1.9.3.1)] based upon redox linkage at the "high-potential" CuB center. A model is proposed that is derived from a redox-linked ligand exchange mechanism previously described for the CuA site. Qualitative analysis of this mechanism indicates that such a mechanism is feasible. However, the relatively short distance between CuB and cytochrome a3 implies that the uncoupling electron transfers are quite facile. In addition, the position of the CuB center with respect to the inner mitochondrial membrane argues against redox linkage at the CuB site.
Asunto(s)
Cobre/química , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/fisiología , Transporte Biológico Activo , Transporte de Electrón , Concentración de Iones de Hidrógeno , Membranas Intracelulares/fisiología , Ligandos , Potenciales de la Membrana , Modelos Biológicos , Oxidación-ReducciónRESUMEN
p-Hydroxymercuribenzoic acid modification of cytochrome c oxidase converts the CuA center into a type 2 copper site while heat treatment of the oxidase in lauryl maltoside can transform CuA almost stoichiometrically (greater than 90%) to a blue type 1 copper site. These modifications of the protein have previously been shown to have a profound effect on the dioxygen reduction and proton pumping activities of the enzyme (Li, P. M., Morgan, J. E., Nilsson, T., Ma, M., and Chan, S. I. (1988) Biochemistry 27, 7538; Nilsson, T., Gelles, J., Li, P. M., and Chan, S. I. (1988) Biochemistry 27, 296; Sone, N., and Nicholls, P. (1984) Biochemistry 23, 6550). In this work, the intrinsic reduction potentials and the midpoint reduction potentials of the "CuA" site in both these modified oxidases have been measured under various conditions in order to clarify the intramolecular electron transfer pathways in these systems. The study reveals that the CuA intrinsic reduction potential decreases by almost 150 mV upon p-hydroxymercuribenzoic acid modification whereas it increases by 100 mV upon heat treatment. In addition, the redox interactions between CuA and the remaining metal centers are perturbed upon CuA modification. It is argued that these results bear on the role of CuA in the proton-pumping paradigm of cytochrome c oxidase.
Asunto(s)
Cobre/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Hidroximercuribenzoatos/farmacología , Animales , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Calor , Oxidación-ReducciónRESUMEN
The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.
Asunto(s)
Bacillus/metabolismo , Grupo Citocromo c/metabolismo , Animales , Hemo/metabolismo , Caballos , Concentración de Iones de Hidrógeno , Solubilidad , Espectrometría Raman/métodosRESUMEN
Resonance Raman spectroscopy was employed to investigate the heme structures of catalytic intermediates of cytochrome c oxidase at room temperature. The high-frequency resonance Raman spectra were obtained for compound C (the two-electron-reduced dioxygen intermediate), ferryl (the three-electron-reduced dioxygen intermediate), and the fully oxidized enzyme. Compound C was formed by photolyzing CO mixed-valence enzyme in the presence of O2. The ferryl intermediate was formed by reoxidation of the fully reduced enzyme by an excess of H2O2. Two forms of the oxidized enzyme were prepared by reoxidizing the fully reduced enzyme with O2. Our data indicate that, in compound C, cyt a3 is either intermediate or low spin and is nonphotolabile and its oxidation state marker band, v4, appears a higher frequency than that of the resting form of the enzyme. The ferryl intermediate also displays a low-spin cyt a3, which is nonphotolabile, and an even higher frequency for the oxidation state marker band, v4. The reoxidized form of cytochrome c oxidase with a Soret absorption maximum at 420 nm has an oxidation state marker band (v4) in a position similar to that of the resting form, while the spin-state region resembles that of compound C. This species subsequently decays to a second oxidized from of the enzyme, which displays a high-frequency resonance Raman spectrum identical with that of the original resting enzyme.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Oxígeno/metabolismo , Animales , Bovinos , Citocromo-c Peroxidasa/metabolismo , Hemo/química , Peróxido de Hidrógeno/metabolismo , Mitocondrias Cardíacas/enzimología , Oxidación-Reducción , Espectrometría Raman , TemperaturaRESUMEN
Resonance Raman spectra of Chromatium vinosum cytochrome c' have been obtained for the five pH-dependent states of the protein [i.e., types I (pH 7), II (pH 10), and III (pH 12) of the ferric protein and type a (pH 7) and type n (pH 12) of the ferrous protein]. The raman spectra of type II and type a are consistent with those of high-spin, 5-coordinate heme proteins, such as deoxyhemoglobin, while spectra of type III and type n correspond more closely to those of low-spin, ferric and ferrous cytochrome c, respectively. Spectra of the CO-bound equilibrium species qualitatively resemble those of carbon monoxy human HbA. However, both the Fe-C and C = O stretching modes of the ligated species exhibit pH-dependent frequency shifts. Our data also indicate that CO photolysis is much more efficient at pH 7 than at pH 12. Moreover, the spectra of the photolytic transients suggest that unique, high-spin species are formed subsequent to CO photolysis from both type a and type n species.
Asunto(s)
Monóxido de Carbono/análisis , Chromatium/enzimología , Grupo Citocromo c/análisis , Sitios de Unión , Compuestos Férricos/análisis , Compuestos Ferrosos/análisis , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Fotólisis , Conformación Proteica , Espectrometría Raman/métodosRESUMEN
Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.
Asunto(s)
Hemoglobina A , Hemoglobinas , Aspirina/análogos & derivados , Monóxido de Carbono , Reactivos de Enlaces Cruzados , Fluorescencia , Concentración de Iones de Hidrógeno , Fotólisis , Espectrometría Raman , TriptófanoRESUMEN
Modification of the CuA site in mammalian cytochrome c oxidase has been used to elucidate the functional role of this center in the catalytic cycle of the enzyme. Both heat treatment in detergents and chemical modification by p-(hydroxymercuri)benzoate (pHMB) convert CuA to a lower potential type II center and effectively remove the site from the electron-transfer pathway during turnover. In this study, resonance Raman spectroscopy has been employed to investigate the effects of these CuA modifications on the heme active sites. The Raman data indicate some environmental perturbation of the heme a3 chromophore in the modified derivatives. Only pHMB modification and SB-12 heat treatment produced significant effects in the Raman spectra of the fully reduced enzyme. These perturbations are much less evident in the spectra obtained within 10 ns of CO photolysis from the fully reduced species of the modified enzymes. Transient Raman studies further indicate that the half-time for CO religation in the modified enzymes is quite similar to that of the native protein.