Optical Shaping of X-Ray Free-Electron Lasers.

In this Letter we report the experimental demonstration of a new temporal shaping technique for x-ray free-electron lasers (FELs). This technique is based on the use of a spectrally shaped infrared (IR) laser and allows optical control of the x-ray generation process. By accurately manipulating the spectral amplitude and phase of the IR laser, we can selectively modify the electron bunch longitudinal emittance thus controlling the duration of the resulting x-ray pulse down to the femtosecond time scale. Unlike other methods currently in use, optical shaping is directly applicable to the next generation of high-average power x-ray FELs such as the Linac Coherent Light Source-II or the European X-FEL, and it enables pulse shaping of FELs at the highest repetition rates. Furthermore, this laser-shaping technique paves the way for flexible tailoring of complex multicolor FEL pulse patterns required for nonlinear multidimensional x-ray spectroscopy as well as novel multicolor diffraction imaging schemes.

High-intensity double-pulse X-ray free-electron laser.

The X-ray free-electron laser has opened a new era for photon science, improving the X-ray brightness by ten orders of magnitude over previously available sources. Similar to an optical laser, the spectral and temporal structure of the radiation pulses can be tailored to the specific needs of many experiments by accurately manipulating the lasing medium, that is, the electron beam. Here we report the generation of mJ-level two-colour hard X-ray pulses of few femtoseconds duration with an XFEL driven by twin electron bunches at the Linac Coherent Light Source. This performance represents an improvement of over an order of magnitude in peak power over state-of-the-art two-colour XFELs. The unprecedented intensity and temporal coherence of this new two-colour X-ray free-electron laser enable an entirely new set of scientific applications, ranging from X-ray pump/X-ray probe experiments to the imaging of complex biological samples with multiple wavelength anomalous dispersion.

Evidence of high harmonics from echo-enabled harmonic generation for seeding x-ray free electron lasers.

Echo-enabled harmonic generation free electron lasers hold great promise for the generation of fully coherent radiation in x-ray wavelengths. Here we report the first evidence of high harmonics from the echo-enabled harmonic generation technique in the realistic scenario where the laser energy modulation is comparable to the beam slice energy spread. In this experiment, coherent radiation at the seventh harmonic of the second seed laser is generated when the energy modulation amplitude is about 2-3 times the slice energy spread. The experiment confirms the underlying physics of echo-enabled harmonic generation and may have a strong impact on emerging seeded x-ray free electron lasers that are capable of generating laserlike x rays which will advance many areas of science.

Demonstration of the echo-enabled harmonic generation technique for short-wavelength seeded free electron lasers.

We report the first experimental demonstration of the echo-enabled harmonic generation technique, which holds great promise for generation of high-power, fully coherent short-wavelength radiation. In this experiment, coherent radiation at the 3rd and 4th harmonics of the second seed laser is generated from the so-called beam echo effect. The experiment confirms the physics behind this technique and paves the way for applying the echo-enabled harmonic generation technique for seeded x-ray free electron lasers.

Measurements and simulations of ultralow emittance and ultrashort electron beams in the linac coherent light source.

The Linac Coherent Light Source (LCLS) is an x-ray free-electron laser project presently in a commissioning phase at the SLAC National Accelerator Laboratory. We report here on very low-emittance measurements made at low bunch charge, and a few femtosecond bunch length produced by the LCLS bunch compressors. Start-to-end simulations associated with these beam parameters show the possibilities of generating hundreds of GW at 1.5 A x-ray wavelength and nearly a single longitudinally coherent spike at 1.5 nm with 2-fs duration.

[Reconstitution of the cholesterol-hydroxylating system from adrenal cortex mitochondria by cytochrome P-450scc, adrenodoxin and the redox-mediator neutral red]. / Rekonstruktsiia kholesteringidroksiliruiushchei sistemy iz mitokhondrii kory nadpochechnikov na osnove tsitokhroma P-450scc, adrenodoxina i redoks-mediatora neitral'nogo krasnogo.

It has been found that 3-amino-7-dimethylamino-2-methylphenazine (neutral red, NR) is responsible for the electron transport from the cathode to adrenodoxin (Ad) and cytochrome P-450 (P-450scc) from adrenal cortex mitochondria inaccessible to direct electrochemical reduction under native conditions. The rate constant for Ad reduction by this mediator is equal to 1.1 x 10(5) M-1 s-1 at 25 degrees C; the values of enthalpy and entropy for the activation reaction are 26.6 kJ.mol-1 and -59.6 J.mol-1 deg-1, respectively. Using the shunted electron transport chain NR----Ad----P-450scc, the cholesterol conversion into pregnenolone in an electrochemical cell was performed. Pregnenolone was found to be the sole steroid product of this reaction. Superoxide dismutase and catalase had no effect on the activity of the shunted system. After removal or substitution of Ad the apo-Ad hemoprotein was reduced in a non-productive manner. Under identical reconstitution conditions methylviologen was ineffective as an electron carrier.

[Purification and characteristics of highly active NADPH-cytochrome P-450 reductase from liver microsomes of phenobarbital-induced rabbits]. / Ochistka i kharakteristika vysokoaktivnoo NADPH-tsitokhrom-P-450- reduktazy iz mikrosom pecheni krolikov, indutsirovannykh fenobarbitalom.

Liver microsomal NADPH-cytochrome P-450 reductase from phenobarbital-induced rabbits was purified by a simple and reproducible method employing combination of 2',5'-ADP-sepharose affinity chromatography and 1-amino-2-hydroxypropyl-sepharose (ADP-sepharose) ion exchange chromatography. Comparison with traditionally used adsorbents revealed advantages of AHP-sepharose for isolation of highly active enzyme preparations. The enzyme was purified 408-fold with a 92% yield of the total activity. Electrophoretic and spectral properties of the preparation corresponded to those of native flavoprotein. The specific NADPH-cytochrome c reductase activity of the purified enzyme (85.7 U/mg at pH 7.7 and 30 degrees C) was 1.5-2.5 times higher than that previously reported.

[Comparative study of the C27-steroid-hydroxylating system from bovine liver mitochondria]. / Sravnitel'noe izuchenie C27-steroidgidroksiliruiushchei sistemy iz mitokhondrii pecheni byka.

A method for purification of C27-steroid hydroxylating cytochrome P-450 (cytochrome P-450(27)) from bovine liver mitochondria was developed. The purification procedure included enzyme extraction from submitochondrial particles with sodium cholate, ammonium sulfate fractionation and biospecific chromatography on cholate-Sepharose and adrenodoxin-Sepharose. The resulting enzyme preparation (317-fold purification, 16% yield) was not electrophoretically homogeneous but did not contain hemoprotein admixtures. The kinetic parameters of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation in a reconstituted system containing hepatoredoxin reductase, hepatoredoxin and cytochrome P-450(27) (Km = 23 microM, kcat = 0.3 s-1 at 25 degrees C) were determined. A reciprocal functional equivalency of hepatoredoxin reductase and adrenodoxin reductase as well as of hepatoredoxin and adrenodoxin in reconstituted systems of steroid 27-hydroxylation (liver) and cholesterol side chain cleavage (adrenal cortex) was established. This equivalency was thought to be due to the similarity in essential physico-chemical properties of reductase components which was especially well-pronounced in the case of hepatoredoxin and adrenodoxin. Estimation of the functional role of lysine, dicarboxylic acid and histidine residues in ferredoxin molecules by the chemical modification method revealed the similarity of the structural organization of their protein globules: the polar residues were shown to be essential for the maintenance of native conformation; dicarboxylic acid residues formed a binding domain for the interaction with electron transport proteins, whereas histidine residues seem to participate in electron transport. At the same time, cytochrome P-450(27) and cytochrome P-450 which split the side chain of cholesterol differ in their substrate specificity, immunochemical and catalytic properties.

[Various properties of purified hepatoredoxin from bovine liver mitochondria]. / Nekotorye svoistva ochishchennogo gepatoredoksina iz mitokhondrii pecheni byka.

Hepatoredoxin purified to homogeneity from bovine liver mitochondria has been characterized for the first time in terms of its most important physico-chemical properties. The protein was found to contain in its active center a [2Fe-2S] cluster and has in the oxidized state an absorption maxima at 280, 320, 415 and 455 nm. The spectrophotometric index of purity, A415/A280 of the homogeneous native preparation is 0.84; extinction coefficient, epsilon 415, is 9800 M-1cm-1. The Mr of hepatoredoxin as evidenced by data from SDS gel electrophoresis is 12 500 Da; pI is 4.2. Hepatoredoxin is necessary for the reconstitution of the C27-steroid hydroxylase activity and can be substituted for by a related protein, adrenodoxin. All the above parameters as well as the circular dichroism spectra, immunochemical properties and sequence of the initial five N-terminal amino acids of hepatoredoxin and adrenodoxin are either coincident or very close. At the same time, the amino acid composition of these ferredoxins, apart from some common features, has individual peculiarities.

Selectively immobilized cytochrome c as an effective affinity ligand for electron transfer proteins.

Preparations of horse heart cytochrome c have been obtained immobilized on Sepharose derivatives via lysine epsilon-amino groups, carboxyl groups of aspartic and glutamic acid residues, methionine and histidine residues as well as imidazole groups additionally introduced by means of modification of free carboxyl groups by histamine. Dissociation constants have been determined for complexes of adrenodoxin, hepatoredoxin , cytochrome b5 heme-containing tryptic fragment and myoglobin with cytochrome c preparations immobilized via lysine residues (cytochrome c-Sepharose I) or additional imidazole groups (cytochrome c-Sepharose II). The latter adsorbent possesses a 2-3 times higher affinity to adrenodoxin and hepatoredoxin than the former. The parameters of interaction with cytochrome c-Sepharose II constitute for the proteins studied the following sequence: adrenodoxin (the highest affinity) greater than or equal to hepatoredoxin greater than cytochrome b4 heme- containing tryptic fragment greater than myoglobin. The efficiency of cytochrome c-Sepharose II application in the course of adrenodoxin, hepatoredoxin and cytochrome b5 purification, as well as isolation of cytochrome b4 heme-containing tryptic fragment has been shown.

[The C27-steroid hydroxylating system from bovine liver mitochondria. Isolation of ferredoxin (hepatoredoxin) by affinity chromatography on cytochrome-c-sepharose]. / C27-steroidgidroksiliruiushchaia sistema iz mitokhondrii pecheni byka. Vydelenie ferredoksina (gepatoredoksina) affinnoi khromatografiei na tsitokhrom-c-sefaroze.

A technique for the isolation of highly purified hepatoredoxin involving the DE-32 cellulose chromatography of post-mitochondrial supernatant, ammonium-sulfate fractionation, Sephadex G-75 gel chromatography, 1-amino-2-hydroxypropyl-Sepharose ion-exchange chromatography and cytochrome-c-Sepharose affinity chromatography is described. The protein was purified 160-fold with a yield of 19%. The synthesis of cytochrome-c-Sepharose was carried out in a way preventing modification of the lysine-containing binding domain of the cytochrome c molecule. To achieve this, free carboxyl groups were modified with histamine to introduce imidazole residues in cytochrome c and the modified protein was immobilized on bromoacetyl-Sepharose.

[Immobilized cytochrome c--an effective ligand for affinity chromatography of electron transport proteins]. / Immobilizovannyi tsitokhrom c--éffektivnyi ligand dlia affinnoi khromatografii élektrontransportnykh belkov.

Preparations of horse heart cytochrome c have been obtained immobilized on Sepharose derivatives via lysine epsilon-amino groups, carboxyl groups of aspartic and glutamic acid residues, methionine and histidine residues as well as imidazole groups additionally introduced by means of chemical modification of free carboxyl groups by histamine. Dissociation constants have been determined for complexes of adrenodoxin, hepatoredoxin, cytochrome b5 heme-containing fragment and myoglobin with preparations of cytochrome c immobilized via lysine residues (adsorbent I) or additionally introduced imidazole groups (adsorbent II). The latter is found to possess a 2-3 times greater affinity for adrenodoxin and hepatoredoxin than the former. The affinity of the proteins studied for the adsorbent II constitutes the following sequence: adrenodoxin greater than or equal to hepatoredoxin greater than cytochrome b5 heme-containing fragment greater than myoglobin. The adsorbent II is shown to be effective when used for purification of hepatoredoxin, adrenodoxin, cytochrome b5 and isolation of cytochrome b5 heme-containing fragment.