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1.
Proc Natl Acad Sci U S A ; 116(9): 3572-3577, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808749

RESUMO

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Ferro/química , Oxigênio/química , Animais , Catálise , Domínio Catalítico , Bovinos , Cobre/química , Cristalografia por Raios X , Complexo IV da Cadeia de Transporte de Elétrons/genética , Oxirredução , Conformação Proteica
2.
Proc Natl Acad Sci U S A ; 114(30): 8011-8016, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28698372

RESUMO

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Animais , Monóxido de Carbono/metabolismo , Bovinos , Cristalografia por Raios X , Complexo IV da Cadeia de Transporte de Elétrons/química , Conformação Proteica
3.
Nat Commun ; 11(1): 4511, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908128

RESUMO

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.


Assuntos
Cristalografia/instrumentação , Elétrons , Dispositivos Lab-On-A-Chip , Lasers , Aldeído Liases/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Hidrodinâmica
4.
Acta Crystallogr D Struct Biol ; 72(Pt 7): 849-59, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27377382

RESUMO

Second harmonic generation correlation spectroscopy (SHG-CS) is demonstrated as a new approach to protein nanocrystal characterization. A novel line-scanning approach was performed to enable autocorrelation analysis without sample damage from the intense incident beam. An analytical model for autocorrelation was developed, which includes a correction for the optical scattering forces arising when focusing intense, infrared beams. SHG-CS was applied to the analysis of BaTiO3 nanoparticles ranging from 200 to ∼500 nm and of photosystem I nanocrystals. A size distribution was recovered for each sample and compared with the size histogram measured by scanning electron microscopy (SEM). Good agreement was observed between the two independent measurements. The intrinsic selectivity of the second-order nonlinear optical process provides SHG-CS with the ability to distinguish well ordered nanocrystals from conglomerates and amorphous aggregates. Combining the recovered distribution of particle diameters with the histogram of measured SHG intensities provides the inherent hyperpolarizability per unit volume of the SHG-active nanoparticles. Simulations suggest that the SHG activity per unit volume is likely to exhibit relatively low sensitivity to the subtle distortions within the lattice that contribute to resolution loss in X-ray diffraction, but high sensitivity to the presence of multi-domain crystals.


Assuntos
Proteínas de Bactérias/química , Compostos de Bário/química , Cristalização/métodos , Nanopartículas/química , Complexo de Proteína do Fotossistema I/química , Análise Espectral/métodos , Synechococcus/química , Titânio/química , Difusão , Raios Infravermelhos , Microscopia Eletrônica de Varredura , Fenômenos Ópticos
5.
Science ; 352(6286): 725-9, 2016 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-27151871

RESUMO

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efeitos da radiação , Cristalografia , Isomerismo , Luz , Fótons , Conformação Proteica/efeitos da radiação , Fatores de Tempo
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