RESUMEN
The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. The crystal structure of the MauG-preMADH complex revealed the presence of a Ca(2+) in proximity to the two hemes [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394]. This Ca(2+) did not readily dissociate; however, after extensive treatment with EGTA or EDTA MauG was no longer able to catalyze TTQ biosynthesis and exhibited altered absorption and resonance Raman spectra. The changes in spectral features are consistent with Ca(2+)-dependent changes in heme spin state and conformation. Addition of H(2)O(2) to the Ca(2+)-depleted MauG did not yield spectral changes characteristic of formation of the bis-Fe(IV) state which is stabilized in native MauG. After addition of Ca(2+) to the Ca(2+)-depleted MauG, full TTQ biosynthesis activity and reactivity toward H(2)O(2) were restored, and the spectral properties returned to those of native MauG. Kinetic and equilibrium studies of Ca(2+) binding to Ca(2+)-depleted MauG indicated a two-step mechanism. Ca(2+) initially reversibly binds to Ca(2+)-depleted MauG (K(d) = 22.4 µM) and is followed by a relatively slow (k = 1.4 × 10(-3) s(-1)) but highly favorable (K(eq) = 4.2) conformational change, yielding an equilibrium dissociation constant K(d,eq) value of 5.3 µM. The circular dichroism spectra of native and Ca(2+)-depleted MauG were essentially the same, consistent with Ca(2+)-induced conformational changes involving domain or loop movements rather than general unfolding or alteration of secondary structure. These results are discussed in the context of the structures of MauG and heme-containing peroxidases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Calcio/metabolismo , Indolquinonas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Paracoccus denitrificans/enzimología , Triptófano/análogos & derivados , Proteínas Bacterianas/química , Sitios de Unión , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Paracoccus denitrificans/química , Paracoccus denitrificans/metabolismo , Estructura Secundaria de Proteína , Espectrofotometría , Espectrometría Raman , Triptófano/metabolismoRESUMEN
The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 Å separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)âO/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)âO heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.
Asunto(s)
Hemo/metabolismo , Histidina/metabolismo , Tirosina , Compuestos Férricos/química , Compuestos Ferrosos/química , Hemo/química , Ligandos , Modelos Moleculares , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Conformación Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Quinolinas/metabolismo , Proteína Smad1 , Espectrometría RamanRESUMEN
Surface-enhanced resonance Raman scattering (SERRS) spectra of biological species are often different from their resonance Raman (RR) spectra. A home-designed Raman flow system is used to determine the factors that contribute to the difference between the SERRS and RR of met-myoglobin (metMb). The results indicate that both the degree of protein-nanoparticles interaction and the laser irradiation contribute to the structural changes and are responsible for the observed differences between the SERRS and RR spectra of metMb. The prolonged adsorption of the protein molecules on the nanoparticle surface, which is the condition normally used for the conventional SERRS experiments, disturbs the heme pocket structure and facilitates the charge transfer process and the photoinduced transformation of proteins. The disruption of the heme pocket results in the loss of the distal water molecule, and the resulting SERRS spectrum of metMb shows a 5-coordinated high-spin heme. The flow system, when operated at a moderately high flow rate, can basically eliminate the factors that disturb the protein structure while maintaining a high enhancement factor. The SERRS spectrum obtained from a 1 x 10 (-7) M metMb solution using this flow system is basically identical to the RR spectrum of a 5 x 10 (-4) M metMb solution. Therefore, the Raman flow system reported here should be useful for characterizing the protein-nanoparticles interaction and the native structure of proteins using SERRS spectroscopy.
Asunto(s)
Metamioglobina/química , Espectrometría Raman/métodos , Adsorción , Animales , Caballos , Nanopartículas del Metal/química , Plata/química , Espectrofotometría UltravioletaRESUMEN
The photocytotoxicity of 16 polycyclic aromatic hydrocarbons (PAHs) on the priority pollutant list of the United States Environmental Protection Agency (US EPA) were tested in human skin HaCaT keratinocytes. A selected PAH was mixed with HaCaT cells and irradiated with a solar simulator lamp for a dose equivalent to 5 min of outdoor sunlight and the cell viability was determined immediately and also after 24 h of incubation. For the cells without incubation after the treatments, it is found that all PAHs with three rings or less, except anthracene, are not photocytotoxic, while the four or five-ring PAHs (except chrysene), benz[a]anthracene, dibenzo[a,h]anthracene, benzo[ghi]perylene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene, benzo[b]fluorenthene, fluorenthene, and pyrene, are photocytotoxic to the human skin HaCaT keratinocytes. If the cells were incubated for 24 h after the treatments, the photocytotoxic effect of the PAHs was greatly amplified in comparison to the nonincubated cells. For the 24 h incubated cells, all PAHs except naphthalene exhibit photocytotoxicity to some extent. Exposure to 5 microM of the 4- and 5-ring PAHs (except chrysene) and 3-ring anthracene more than 80% of the cells lose viability. The photocytotoxicity of the PAHs correlates well with several of their excited state properties: light absorption, excited singlet-state energy, excited triplet-state energy, and HOMO-LUMO energy gap. All the photocytotoxic PAHs absorb light at >300 nm, in the solar UVB and UVA region. There is a threshold for each of the three excited state descriptors of a photocytotoxic PAH: singlet energy <355 kJ/mol (corresponding to 337 nm light), triplet energy <230 kJ/mol (corresponding to 520 nm light), HOMO-LUMO gap <3.6 eV (corresponding to 344 nm light) obtained at the Density Functional Theory B3LYP/6-31G(d) level.
Asunto(s)
Contaminantes Ambientales/toxicidad , Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiación , Hidrocarburos Policíclicos Aromáticos/toxicidad , Rayos Ultravioleta , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Contaminantes Ambientales/química , Humanos , Estructura Molecular , Pruebas de Mutagenicidad , Mutágenos/química , Mutágenos/toxicidad , Hidrocarburos Policíclicos Aromáticos/química , Piel/efectos de los fármacos , Piel/efectos de la radiación , Factores de Tiempo , Pruebas de Toxicidad/métodos , Estados Unidos , United States Environmental Protection AgencyRESUMEN
Layer-by-layer assembly of glucose oxidase (GOx) with single-wall carbon nanotubes (SWCNTs) is achieved on the electrode surface based on the electrostatic attraction between positively charged GOx in pH 3.8 buffer and negatively charged carboxylic groups of CNTs. The cyclic voltammetry and electrochemical impedance spectroscopy are used to characterize the formation of multilayer films. In deaerated buffer solutions, the cyclic voltammetry of the multilayer films of {GOx/CNT}(n) shows two pairs of well-behaved redox peaks that are assigned to the redox reactions of CNTs and GOx, respectively, confirming the effective immobilization of GOx on CNTs using the layer-by-layer technique. The redox peak currents of GOx increase linearly with the increased number of layers indicating the uniform growth of GOx in multilayer films. The dependence of the cyclic voltammetric response of GOx in multilayer films on the scan rate and pH is also studied. A linear decrease of the reduction current of oxygen at the {GOx/CNT}-modified electrodes with the addition of glucose suggests that such multilayer films of GOx retain the bioactivity and can be used as reagentless glucose biosensors.
Asunto(s)
Técnicas Biosensibles/instrumentación , Glucosa Oxidasa/química , Glucosa/análisis , Nanotubos de Carbono/química , Técnicas Biosensibles/métodos , Catálisis , ElectroquímicaRESUMEN
MauG is a novel 42 kDa diheme protein which is required for the biosynthesis of tryptophan tryptophylquinone, the prosthetic group of methylamine dehydrogenase. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes and the overall redox properties of MauG are described. The absorption maxima for the Soret peaks of the oxidized and reduced hemes are 403 and 418 nm for the low-spin heme and 389 and 427 nm for the high-spin heme, respectively. The resonance Raman spectrum of oxidized MauG exhibits a set of marker bands at 1503 and 1588 cm(-1) which exhibit frequencies similar to those of the nu3 and nu2 bands of c-type heme proteins with bis-histidine coordination. Another set of marker bands at 1478 and 1570 cm(-1) is characteristic of a high-spin heme. Two distinct oxidation-reduction midpoint potential (E(m)) values of -159 and -244 mV are obtained from spectrochemical titration of MauG. However, the two nu3 bands located at 1478 and 1503 cm(-1) shift together to 1467 and 1492 cm(-1), respectively, upon reduction, as do the Soret peaks of the low- and high-spin hemes in the absorption spectrum. Thus, the two hemes with distinct spectral properties are reduced and oxidized to approximately the same extent during redox titrations. This indicates that the high- and low-spin hemes have similar intrinsic E(m) values but exhibit negative redox cooperativity. After the first one-electron reduction of MauG, the electron equilibrates between hemes. This makes the second one-electron reduction of MauG more difficult. Thus, the two E(m) values do not describe redox properties of distinct hemes, but the first and second one-electron reductions of a diheme system with two equivalent hemes. The structural and mechanistic implications of these findings are discussed.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hemo/metabolismo , Indolquinonas/biosíntesis , Oxígeno/metabolismo , Triptófano/análogos & derivados , Electroquímica , Hemo/clasificación , Cinética , Modelos Moleculares , Oxidación-Reducción , Paracoccus denitrificans , Triptófano/biosíntesisRESUMEN
The direct electrochemistry of myoglobin (Mb) has been observed at a glassy carbon (GC) electrode coated with silica sol-gel-encapsulated Mb film. A well-behaved cyclic voltammogram is observed with a midpoint potential (E(1/2)) of -0.25 V vs Ag/AgCl in a pH 7.0 phosphate buffer. This potential, which is pH-dependent, is 70-90 mV more negative than the formal potential values obtained by using the spectroeletrochemical titration method at the same pH. Square wave voltametry (SWV) also shows a peak potential of -0.25 V for the reduction of Mb under the same experimental conditions. Both cathodic and anodic peak currents have a linear relationship with the scan rate. The midpoint potential decreases with pH, having a slope of -30 mV/pH. UV-vis and resonance Raman spectroscopic studies reveal that the sol-gel provides a bio-compatible environment where Mb retains a structure similar to its solution form, a 6-coordinated aquomet myoglobin. These results suggest that the silica sol-gel is a useful matrix for studying direct electrochemistry of other heme proteins.
Asunto(s)
Electroquímica/métodos , Mioglobina/química , Espectrometría Raman/métodos , Animales , Electrodos , Hemo/química , Concentración de Iones de Hidrógeno , Transición de Fase , Fosfatos/química , Rayos UltravioletaRESUMEN
The electrocatalytic oxidation of thiols has been observed at a glassy carbon (GC) electrode coated with a single-wall carbon nanotube (SWNT) film. Fourteen thiols including L-cysteine (CySH) and glutathione were tested using the SWNT/GC electrode, and the cyclic voltammetry (CV) showed that each thiol was oxidized at much less positive potential than those at other electrodes such as bare GC and diamond electrodes. The SWNT/GC electrode was also modified with pyrroloquinoline quinone (PQQ) which showed a further improvement of the catalytic behavior of the SWNT/GC electrode: e.g. the oxidation peak current of CySH was observed at 0.27 V vs. Ag/AgCl in pH 7.5 phosphate buffer. The amperometic responses at these electrodes showed a linear relationship with the substrate concentration in a 10(-6)-10(-3) M range and 10(-6)-10(-7) M detection limits for several thiols including CySH, L-homocysteine, N-acetyl-L-cysteine, L-penicillamine and glutathione. These electrodes show a response time of 2-3 s and storage stabilities over 3 weeks. A PQQ/SWNT/GC electrode has been successfully applied for the assay of both L-cysteine and N-acetyl-L-cysteine in the dietary supplement.
Asunto(s)
Cisteína/análisis , Electroquímica/métodos , Nanotubos de Carbono , Cofactor PQQ/química , Compuestos de Sulfhidrilo/análisis , Electrodos , Modelos Químicos , Compuestos de Sulfhidrilo/químicaRESUMEN
Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP [C cP(MI, G41E, V45E, H181D)], MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).
Asunto(s)
Citocromo-c Peroxidasa/química , Citocromo-c Peroxidasa/genética , Peroxidasas/metabolismo , Espectrometría Raman , Sustitución de Aminoácidos , Sitios de Unión , Citocromo-c Peroxidasa/metabolismo , Proteínas Fúngicas , Hemo/química , Peróxido de Hidrógeno/metabolismo , Cinética , Imitación Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de ProteínasRESUMEN
We have evaluated the use of cytochrome P450 (CYP), 3A4 genotype and Fourier transform-infrared (RT-IR)/Raman spectroscopy as diagnostic tools for detection of breast tumors. CYP is involved in catalytic activity of oxidative metabolism of many chemicals in fatty tissues, and it plays a major role in biotransformation and detoxication of environmental contaminants. FT-IR and Raman spectroscopy have been used to develop methods for cancer assessment. Thus, the hypothesis was that a) CYP 3A4 gene expression level may have effect on the clinical presentation of breast cancer; and b) a combination spectroscopy and genotype analysis may strengthen the level of diagnosis. In parallel studies we compared by reverse-transcription-polymerase chain reaction (RT-PCR), the CYP 3A4 mRNA transcript levels, and by FT-IR the pathology of breast tissues. RNA was isolated from human breast biopsies and cultured tumor cells (MCF-7). A comparison of the levels of RT-PCR was made between CYP 3A4 genotype and 1B1, a genotype associated with human tumors, testing 3 normal breast tissues, 2 specimen from breast reduction and 7 breast tumors. Two variants of CYP 3A4 mRNA were observed, of which a 380-bp was displayed in 4 out of 5 pathologically determined tumors, and a 260-bp fragment was associated with normal tissues. The predictive value of the CYP 3A4 for the detection of tumor tissues was greater than that observed with the CYP 1B1. FT-IR signal patterns were distinct for tumor tissues as compared with that of normal tissue. Our findings demonstrated the importance of CYP 3A4 as molecular biomarker for determining the presence of breast tumors. This data in association with FT-IR/Raman spectroscopy and pathology, it can be an ideal test for predicting the clinical presentation of breast cancer.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría Raman/métodos , Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/química , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Regulación Neoplásica de la Expresión Génica , Genotipo , Humanos , Neoplasias Mamarias Animales/química , Neoplasias Mamarias Animales/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Células Tumorales CultivadasRESUMEN
The heme active site structure of chloroperoxidase (CPO), a glycoprotein that displays versatile catalytic activities isolated from the marine mold Caldariomyces fumago, has been characterized by two-dimensional NMR spectroscopic studies. All hyperfine shifted resonances from the heme pocket as well as resonances from catalytically relevant amino acid residues including the heme iron ligand (Cys(29)) attributable to the unique catalytic properties of CPO have been firmly assigned through (a) measurement of nuclear Overhauser effect connectivities, (b) prediction of the Curie intercepts from both one- and two-dimensional variable temperature studies, (c) comparison with assignments made for cyanide derivatives of several well characterized heme proteins such as cytochrome c peroxidase, horseradish peroxidase, and manganese peroxidase, and (d) examination of the crystal structural parameters of CPO. The location of protein modification that differentiates the signatures of the two isozymes of CPO has been postulated. The function of the distal histidine (His(105)) in modulating the catalytic activities of CPO is proposed based on the unique arrangement of this residue within the heme cavity. Contrary to the crystal state, the high affinity Mn(II) binding site in CPO (in solution) is not accessible to externally added Mn(II). The results presented here provide a reasonable explanation for the discrepancies in the literature between spectroscopists and crystallographers concerning the manganese binding site in this unique protein. Our study indicates that results from NMR investigations of the protein in solution can complement the results revealed by x-ray diffraction studies of the crystal form and thus provide a complete and better understanding of the actual structure of the protein.
Asunto(s)
Cloruro Peroxidasa/química , Proteínas Fúngicas/química , Hemo/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
Methylamine dehydrogenase (MADH) may be immobilized in a polypyrrole (PPy) film on an electrode surface and used as an amperometric sensor for the determination of histamine. Using site-directed mutagenesis, phenylalanine 55 on the alpha subunit of MADH was converted to alanine. This alphaF55A MADH exhibits a 400-fold lower Km value for histamine than does native MADH when assayed in solution. An alphaF55A MADH-PPy sensor was constructed, and its properties were compared to that of the native MADH-PPy sensor. The alphaF55A MADH immobilized on the electrode exhibited Michaelis-Menten behavior in response to varied concentrations of histamine with an approximately 3-fold lower Km value than that exhibited by the immobilized native MADH. The detection limit for the native MADH-PPy sensor was approximately 20 microM while the alphaF55A MADH-PPy sensor exhibited a detection limit of approximately 5 microM, a 4-fold increase compared to the native MADH-PPy sensor. This work highlights the potential value of using site-directed mutagenesis to engineer enzymes to alter and improve biosensor performance.