RESUMEN
Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 x 100 x 100 microm, belonged to space group P4(3)2(1)2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 A resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C(380), revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.
Asunto(s)
Bacterioclorofilas/química , Hyphomicrobiaceae/química , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Citocromos/química , Oxidación-Reducción , Fenilalanina/química , Feofitinas/química , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Multimerización de Proteína , Espectrofotometría , Tirosina/químicaRESUMEN
The chemistry of bacterial photosynthesis begins in the photosynthetic reaction centre (RC), a protein complex containing a series of electron donor and acceptor molecules. Although the pigments of the RC can absorb light to operate the photochemistry, the bulk of the light is captured in special pigmented proteins, the light harvesting complexes (LHCs), that then transfer the energy to the RC. Ordinarily, the LHCs do not participate in chemical reactions during photosynthesis such that LHCs do not become oxidised upon light irradiation. However, upon chemical oxidation in the dark, cation radicals of bacteriochlorophyll (BChl) can be formed in the light harvesting complex 1 (LH1) of Rhodobacter sphaeroides. As observed by continuous-wave electron-paramagnetic resonance (EPR), the charges of the BChl(+) cations migrate rather freely about the LH1 complex as in a molecular wire. Remarkably, these LH1 molecular wires continue to function in the frozen, solid state. To investigate the nature of electron-hole transfer and to corroborate the process as revealed by EPR, electron-nuclear double resonance (ENDOR) was recorded at 80 K. ENDOR observed only monomeric bacteriochlorophyll cations. Their signal intensity decreased with increased oxidation while the EPR signal narrowed and increased in size. At the increased oxidation state, the possibility of spin-spin exchange between two BChl(+)s within LH1 versus electron-hole transfer is addressed. An energy landscape of the BChl(+)s in the LH1 is proposed to explain the EPR and ENDOR results.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón , Complejos de Proteína Captadores de Luz/química , Rhodobacter sphaeroides/enzimología , Transporte de Electrón , Oxidación-ReducciónRESUMEN
Time-resolved electron paramagnetic resonance was used to monitor the photochemistry of radical pairs from melanin in porcine retinal pigment epithelial cells on the sub-microsecond time scale. Two distinct signals were found: one of enhanced absorption/emission at early times and one mostly emissive at later times. The emissive character of the longer lived feature suggests participation of an excited triplet precursor, something not generally thought to exist in melanins. The radicals in the early time signal were separated by about 21 A and those in the later time signal were separated by about 22-24 A.
Asunto(s)
Melaninas/química , Procesos Fotoquímicos , Animales , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Radicales Libres/metabolismo , Melaninas/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Porcinos , Factores de TiempoRESUMEN
Photoactivation in crystals of the bacterial reaction center of Blastochloris viridis was investigated by near-infrared spectroscopy. The bleaching of the special pair absorption at 970 nm and the simultaneous rise of the special pair cation absorption at 1300 nm were measured in response to transient irradiation by a HeNe laser over 5 orders of magnitude in laser power. The resulting power-saturation curve can be used to estimate the true extent of photoactivation achieved in a prior time-resolved crystallographic experiment (Baxter et al. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5982-5987). The overall extent of photoactivation was 50%, which demonstrates that the time-resolved crystallographic method can be applied to the optically dense reaction center crystals. Measurement of the charge-recombination rate, however, suggests the presence of a long-lived P+ state within the crystal.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Proteobacteria/efectos de la radiación , Cristalización , Cristalografía , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/fisiología , Proteobacteria/química , Proteobacteria/fisiología , Espectroscopía Infrarroja CortaRESUMEN
The utility of photosynthetically defective mutants in the purple photosynthetic bacterium Blastochloris viridis (formerly Rhodopseudomonas viridis)was demonstrated with construction of a reaction-center deficient mutant, LH 1-H. This LH 1-H mutant has a photosynthetic apparatus in which most of the puf operon genes were deleted, resulting in an organism containing only the genes for the light harvesting polypeptides and the H subunit of the reaction center. This B. viridisstrain containing a truncation of the puf operon was characterized by gel electrophoresis, lipid-to-protein ratio analysis, optical spectroscopy, electron paramagnetic resonance and transmission electron microscopy. Optical and electron paramagnetic resonance spectroscopies revealed no photoactivity in this LH 1-H mutant consistent with the absence of intact reaction centers. Electron paramagnetic resonance evidence for assembled LH 1 complexes suggested that the interactions between light harvesting polypeptide complexes in membranes were largely unchanged despite the absence of their companion reaction center cores. The observed increase in the lipid-to-protein ratio was consistent with modified interactions between LH 1s, a view supported by transmission electron microscopy analysis of membrane fragments. The results show that B. viridis can serve as a practical system for investigating structure-function relationships in membranes and photosynthesis through the construction of photosynthetically defective mutants.
RESUMEN
We report on the analysis of the inter-bacteriochlorophyll a (BChla) charge-transport process that occurs in oxidized purple bacterial light-harvesting 1 (LH1) complexes. Experimentally, charge migration within oxidized LH1 is monitored by following the temperature-dependent changes of the BChla(â¢)(+) electron paramagnetic resonance (EPR) line-shape characteristics. At 6 K, a Gaussian-shaped spectrum with a 1.3-mT width is detected. These characteristics indicate that at extremely low temperatures charge transport is substantially slowed so that the unpaired electron is localized on one or two BChlas. At higher temperatures, the spectra exhibit non-Gaussian line shapes and decreased line widths. These characteristics are engendered by charge migration. We have analyzed the temperature dependence of the transport process through EPR spectral simulations. The simulations incorporated a nonadiabatic model for electron transfer. The temperature dependence could be adequately described on the basis of an electron-transfer model that accounts for the effects of slow medium relaxation, whereas a satisfactory description could not be obtained on the basis of conventional multimode models for transport. The results of our analysis are consistent with the notion that the protein functions as the primary solvent for the redox centers and are in accord with the view that the protein behaves as a frozen glass, even at room temperature, with respect to the low-frequency vibrational motions coupled to electron transfer.
RESUMEN
Cation free radicals of bacteriochlorophyll (BChl(+)) are formed in the light harvesting complex 1 (LH1) of photosynthetic bacteria upon oxidation by potassium ferricyanide. Unusually narrow EPR line widths are observed for BChl(+) in the frozen state. These narrow line widths are consistent with a molecular-wire behavior where rapid electron/hole transfer occurs between the BChl constituents of the pigment array responsible for light harvesting in bacterial photosynthesis. However, in addition to electron/hole transfer, two distinct types of spin-spin exchange could contribute the EPR line width narrowing, thus obfuscating the determination of LH1 as a molecular wire. First, because excess ferricyanide ion is always present during the EPR measurements, electron spin-spin interactions between the paramagnetic ferricyanide and BChl(+) could be a major source of the EPR line width changes previously attributed solely to electron/hole hopping within the array of BChl molecules in a LH1 unit. Fixing the potential of the ferricyanide/ferrocyanide redox couple gives a constant concentration of paramagnetic iron as the amount of BChl oxidized in LH1 changes. As long as the fraction of oxidized BChl in LH1 remains the same, the EPR line width is found independent of the concentration of the ferricyanide oxidant. Additionally, the trend in EPR line width as a function of temperatures depends only on the fraction of oxidized BChl and not on the concentration of ferricyanide ion. Second, spin-spin exchange interactions between BChl(+)s within LH1 rings could also change the EPR line width. Using LH1 preparations containing at most a few BChl cations per LH1 complex also eliminates the occurrence of significant electron spin-spin exchange as a cause of the observed line width narrowing in minimally oxidized LH1. This investigation of the two types of electron spin-spin exchange interactions demonstrates (1) that electron/hole hopping can take place in oxidized LH1 without involvement of paramagnetic ferricyanide or spin-spin exchange between BChl(+)s and (2) that LH1 maintains a molecular-wire nature at cryogenic temperatures.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterioclorofilas/metabolismo , Congelación , Hierro/química , Complejos de Proteína Captadores de Luz/metabolismo , Magnetismo , Proteínas Bacterianas/química , Bacterioclorofilas/química , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Ferricianuros/química , Ferricianuros/metabolismo , Ferrocianuros/química , Ferrocianuros/metabolismo , Radicales Libres/química , Radicales Libres/metabolismo , Hierro/metabolismo , Complejos de Proteína Captadores de Luz/química , Rhodobacter sphaeroides , TemperaturaRESUMEN
We have designed a protease-sensitive imaging probe for optoacoustic imaging whose absorption spectrum changes upon cleavage by a protease of interest. The probe comprises an active site, a derivative of chlorophyll or natural photosynthetic bacteriochlorophyll that absorbs in the near infrared, conjugated to a peptide backbone specific to the protease being imaged. The uncleaved molecules tend to aggregate in dimers and trimers, causing a change in the absorption spectrum relative to that of the monomer. Upon cleavage, the probe molecules deaggregate, giving rise to a spectrum characteristic of monomers. We show using photospectrometry that the two forms of the probe have markedly different absorption spectra, which could allow for in vivo optoacoustic identification using a multiwavelength imaging strategy. Optoacoustic measurements using a narrow-band dye laser find spectral peaks in the two forms of the probe at the expected location. The optoacoustic signal from the uncleaved probe is found to be considerably weaker than that of the cleaved probe, perhaps due to poor optical-acoustic coupling in the aggregated molecules. However, ultimately, it is detection of the cleaved probe that is of the greatest import, since it reports on the protease activity of interest.
Asunto(s)
Clorofila/química , Medios de Contraste/química , Diagnóstico por Imagen de Elasticidad/métodos , Técnicas de Sonda Molecular , Péptido Hidrolasas/análisis , Activación EnzimáticaRESUMEN
Blue-light excitation of cryptochromes and homologues uniformly triggers electron transfer (ET) from the protein surface to the flavin adenine dinucleotide (FAD) cofactor. A cascade of three conserved tryptophan residues has been considered to be critically involved in this photoreaction. If the FAD is initially in its fully oxidized (diamagnetic) redox state, light-induced ET via the tryptophan triad generates a series of short-lived spin-correlated radical pairs comprising an FAD radical and a tryptophan radical. Coupled doublet-pair species of this type have been proposed as the basis, for example, of a biological magnetic compass in migratory birds, and were found critical for some cryptochrome functions in vivo. In this contribution, a cryptochrome-like protein (CRYD) derived from Xenopus laevis has been examined as a representative system. The terminal radical-pair state FAD(â¢)···W324(â¢) of X. laevis CRYD has been characterized in detail by time-resolved electron-paramagnetic resonance (TREPR) at X-band microwave frequency (9.68 GHz) and magnetic fields around 345 mT, and at Q-band (34.08 GHz) at around 1215 mT. Different precursor states, singlet versus triplet, of radical-pair formation have been considered in spectral simulations of the experimental electron-spin polarized TREPR signals. Conclusively, we present evidence for a singlet-state precursor of FAD(â¢)···W324(â¢) radical-pair generation because at both magnetic fields, where radical pairs were studied by TREPR, net-zero electron-spin polarization has been detected. Neither a spin-polarized triplet precursor nor a triplet at thermal equilibrium can explain such an electron-spin polarization. It turns out that a two-microwave-frequency TREPR approach is essential to draw conclusions on the nature of the precursor electronic states in light-induced spin-correlated radical pair formations.
Asunto(s)
Criptocromos/química , Criptocromos/metabolismo , Luz , Animales , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón/efectos de la radiación , Flavina-Adenina Dinucleótido/metabolismo , Radicales Libres/química , Radicales Libres/metabolismo , Modelos Moleculares , Conformación Proteica , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
PURPOSE: The effect of AcrySof filter (UV light-filtering chromophore; Alcon) and AcrySof Natural filter (UV- and blue light-filtering chromophores) on blue light-induced apoptosis in human retinal pigment epithelial (RPE) cells was evaluated. DESIGN: Laboratory investigation CLINICAL RELEVANCE: Acrysof Natural filter reduces the blue-light toxicity in RPE cells and may have a positive impact on age-related macular degeneration (AMD). METHODS: RPE cells were exposed to blue light (430-450 nm) in the presence of either the AcrySof (UV only) filter or Acrysof Natural (UV and blue light) filter for 10 days. The rate of apoptosis was analyzed. RESULTS: Blue light induced significant apoptosis in RPE cells. AcrySof Natural filter significantly reduced the blue light-induced apoptosis when compared to AcrySof filter. The amount of blue-light energy reaching the cells with the AcrySof filter was 4.25 mW/cm(2) and with the AcrySof Natural filter was 2.5 mW/cm(2). CONCLUSIONS: AcrySof Natural filter significantly reduced blue light-induced apoptosis. This was most likely due to its filtering effect on blue wavelength light, which reduces the energy that reaches the cells. In patients with cataract who are at a high risk for AMD, the implantation of a blue light-filtering intraocular lens may be considered.
Asunto(s)
Apoptosis/efectos de la radiación , Lentes Intraoculares , Luz , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de la radiación , Protección Radiológica/instrumentación , Resinas Acrílicas , Anexina A5/metabolismo , Recuento de Células , Línea Celular , Supervivencia Celular/fisiología , Citoprotección , Filtración/instrumentación , Citometría de Flujo , Humanos , Lipofuscina/metabolismo , Estrés Oxidativo , Epitelio Pigmentado Ocular/metabolismo , Compuestos de Piridinio/metabolismo , Retinoides/metabolismoRESUMEN
As the Fourier transform of time-series data is known as the spectrum, the Fourier transform of the logarithm of the time-series data is called the cepstrum of the data. When cepstral analysis is applied to free induction decay signals of free radicals showing first-order EPR spectra, the identification of nuclear hyperfine coupling constants becomes simple. In a systematic manner, we have examined how the technique of cepstral analysis is affected by the presence of aliasing, noise, uncertainty in the time origin of the free induction decay, the presence of second-order hyperfine couplings, and the applications of various apodization methods. This technique was then applied to analyze the EPR spectrum of anthraquinone anion radical, and anion radicals of porphycene and tetrapropyl-porphycene, and the hyperfine coupling constants thus obtained were compared with published data. A good agreement was always found. We make a case for the usefulness of cepstral analysis in determining the hyperfine coupling constants of complex EPR spectra of organic free radicals.
Asunto(s)
Algoritmos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Dinámicas no Lineales , Compuestos Orgánicos/química , Análisis de Fourier , CinéticaRESUMEN
Photoinduced, proton-coupled electron transfer (ET) between 9,10-anthraquinone-2,6-disulfonate (ADQS) and an amino acid residue of tryptophan in human serum albumin (HSA) was observed using time-resolved electron paramagnetic resonance (TREPR). The ET reaction reduces the protein binding affinity of the ligand. TREPR chemically induced dynamic electron polarization (CIDEP) spectra establish that photoinduced ET takes place from the tryptophan residue (W214) to the excited triplet state of AQDS2- while bound in subdomain IIA, a protein cleft of HSA. The TREPR CIDEP signals also reveal that the anion radical of the ligand escapes toward the bulk water region by a one-dimensional translation diffusion process within the protein's pocket area. This pilot study of HSA demonstrates how TREPR CIDEP can provide significant means to investigate dynamic characteristics of protein-surface reactions.
Asunto(s)
Antraquinonas/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Albúmina Sérica/química , Simulación por Computador , Electrones , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Albúmina Sérica/metabolismo , Propiedades de Superficie , Triptófano/química , Warfarina/químicaRESUMEN
Cultured retinal pigment epithelium (RPE) cells can phagocytize large foreign particles. Heterogeneous melanin aggregates from Sepia officinalis, a species of cuttlefish, were fed to cultured human RPE cells to produce cells laden with Sepia melanin. Blue light-induced apoptosis (BLIA) assays were performed by flow cytometry on parallel cultures consisting of RPE cells isolated from independent eyes and evenly divided into two cultures, one fed Sepia melanin and one containing only native melanin. After culturing and growth of the cells under blue light illumination for 7 days, the apoptosis percentage of all cultures indicated that Sepia feeding significantly reduced BLIA. To account for Sepia photoprotection, continuous-wave EPR and time-resolved EPR experiments were performed with parallel RPE cultures by using UV (355 nm) and green (532 nm) laser irradiation. Continuous-wave EPR spectra prove that the concentrations of intrinsic and extrinsic melanin free radicals in the Sepia-RPE culture are large compared with those concentrations in the RPE culture. Time-resolved EPR spectra indicate that both UV and green light produced extrinsic melanin radicals as radical pairs from the triplet manifold with a linear dependence on the number of photons per second. These experiments conclusively demonstrate that decreased RPE susceptibility to BLIA correlates with increased intracellular melanin free radical concentrations and that nonnative melanin can supplement native melanin photoprotection of RPE cells.
Asunto(s)
Melaninas/metabolismo , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Citometría de Flujo , Radicales Libres , Humanos , Melaninas/química , Melaninas/farmacología , Fagocitosis , Fotobiología , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/efectos de la radiación , Sepia/metabolismoRESUMEN
The structure of the Blastochloris viridis photosynthetic reaction center has been determined at 100 K by flash-freezing crystals. A data set to 2.2 A resolution provides a well determined model of the wild-type protein. Of particular interest are the position, occupancy and heterogeneity of the Q(B)-binding site. Data were also collected from a crystal frozen immediately after illumination. The data support predominant binding of Q(B) in the proximal position in both the neutral and charge-separated states.
Asunto(s)
Fotosíntesis/fisiología , Proteobacteria/metabolismo , Sitios de Unión , Crioprotectores/química , Cristalización , Oscuridad , Congelación , Herbicidas/farmacología , Luz , Modelos Moleculares , Quinonas/química , Ubiquinona/metabolismo , Agua/químicaRESUMEN
Photoinduced primary charge-separation and charge-recombination are characterized by a combination of time-resolved optical and EPR measurements of a fullerene-porphyrin-linked triad that undergoes fast, stepwise charge-separation processes. The electronic coupling for the energy-wasting charge recombination is evaluated from the singlet-triplet electronic energy gap in the short-lived, primary charge-separated state. The electronic coupling is found to be smaller by approximately 40% than that for the primary charge-separation. This inhibition of the electronic interaction for the charge-recombination to excited triplet state largely results from a symmetry-broken electronic structure modulated by configuration interaction between 3(b1u,b3g) and 3(au, b3g) electronic states of the free-base porphyrin.
Asunto(s)
Electrones , Fulerenos/química , Modelos Químicos , Nanotecnología , Fotoquímica/métodos , Proteínas del Complejo del Centro de Reacción Fotosintética , Porfirinas/química , Espectrometría de FluorescenciaRESUMEN
Melanin, a ubiquitous, heterogeneous biological polymer composed of many different monomers, contains a population of stationary, intrinsic semiquinone-like radicals. Additional extrinsic semiquinone-like radicals are reversibly photogenerated with visible or UV irradiation. The free radical chemistry of melanin is complex and not well characterized, especially the photochemistry of melanin in the presence of oxygen. To determine directly how melanin reacts in the presence of oxygen, time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to examine melanin free radical chemistry in human retinal pigment epithelium (RPE) cells under aerobic and anaerobic conditions. A TREPR difference spectrum was used to explore the nature of melanin chemistry in the presence of oxygen. The position and symmetrical line shape of the TREPR three-dimensional difference spectrum shows that when reactive oxygen species (ROS) are scavenged, only one of the two or more chemically different melanin free radical species participates in ROS scavenging. This protective melanin radical species exists in both the extrinsic and intrinsic populations of melanin free radicals, allowing melanin to protect the RPE from toxic species in both the light and dark.
Asunto(s)
Melaninas/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Humanos , Melaninas/análisis , Estrés Oxidativo , Epitelio Pigmentado Ocular/químicaRESUMEN
Time-resolved electron paramagnetic resonance (TREPR) spectroscopy was used to study melanin free radicals in human retinal pigment epithelium (RPE) cells and tyrosine-derived synthetic melanin. TREPR signal traces from RPE cells reveal in vivo light-induced melanin free radical photochemistry in more detail than previously known. Electron spin polarization reflecting a non-Boltzmann population within the energy levels of the spin system is observed in RPE cells as the result of the triplet state photoproduction and subsequent disappearance of free radicals in the melanin polymer. In a set of RPE cells cultured from individual sources, differences in optical absorption, continuous wave EPR spectra, and TREPR signals were correlated with apoptosis assays performed by flow cytometry. Continuous wave EPR spectra of RPE cells and TREPR of acidified synthetic melanin suggest that increased melanin aggregation provides an increase in photoprotection in the RPE cells that are relatively less susceptible to blue light-induced apoptosis.
Asunto(s)
Apoptosis/efectos de la radiación , Luz , Melaninas/fisiología , Epitelio Pigmentado Ocular/fisiología , Protección Radiológica , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Feto , Humanos , Melaninas/farmacología , Epitelio Pigmentado Ocular/citología , Epitelio Pigmentado Ocular/efectos de la radiaciónRESUMEN
The photosynthetic reaction center of the purple non-sulfur bacterium Blastochloris viridis was frozen in the presence and absence of illumination. Differences in the resulting datasets are monitored using the difference Fourier method. Radiation damage is localized to those parts of the protein that are significant for electron transfer, and show changes that are sensitive to oxidation and protonation state.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Alphaproteobacteria/química , Citocromos/química , Análisis de Fourier , Hemo/química , Luz , Modelos Moleculares , Conformación Proteica/efectos de la radiación , Rhodobacter sphaeroides/química , Rayos XRESUMEN
Light-induced structural changes in the bacterial reaction center were studied by a time-resolved crystallographic experiment. Crystals of protein from Blastochloris viridis (formerly Rhodopseudomonas viridis) were reconstituted with ubiquinone and analyzed by monochromatic and Laue diffraction, in the dark and 3 ms after illuminating the crystal with a pulsed laser (630 nm, 3 mJ/pulse, 7 ns duration). Refinement of monochromatic data shows that ubiquinone binds only in the "proximal" Q(B) binding site. No significant structural difference was observed between the light and dark datasets; in particular, no quinone motion was detected. This result may be reconciled with previous studies by postulating equilibration of the "distal" and "proximal" binding sites upon extended dark adaption, and in which movement of ubiquinone is not the conformational gate for the first electron transfer between Q(A) and Q(B).