Structure of the cell envelope of Halobacterium halobium

The structure of the isolated cell envelope of Halobacterium halobium is studied by X-ray diffraction, electron microscopy, and biochemical analysis. The envelope consists of the cell membrane and two layers of protein outside. The outer layer of protein shows a regular arrangement of the protein or glycoprotein particles and is therefore identified as the cell wall. Just outside the cell membrane is a 20 A-thick layer of protein. It is a third structure in the envelope, the function of which may be distinct from that of the cell membrane and the cell wall. This inner layer of protein is separated from the outer protein layer by a 65 Å-wide space which has an electron density very close to that of the suspending medium, and which can be etched after freeze-fracture. The space is tentatively identified as the periplasmic space. At NaCl concentrations below 2.0 M, both protein layers of the envelope disintegrate. Gel filtration and analytical ultracentrifugation of the soluble components from the two protein layers reveal two major bands of protein with apparent mol wt of ~16,000 and 21,000. At the same time, the cell membrane stays essentially intact as long as the Mg++ concentration is kept at ≥ 20 mM. The cell membrane breaks into small fragments when treated with 0.1 M NaCl and EDTA, or with distilled water, and some soluble proteins, including flavins and cytochromes, are released. The cell membrane apparently has an asymmetric core of the lipid bilayer

Femtosecond spectroscopy of the first events of the photochemical cycle in bacteriorhodopsin

The first steps in the photochemistry of bacteriorhodopsin (BR) are investigated with light pulses of 160 fs duration. Four samples are studied: (i) the purple membrane, (ii) deuterated purple membrane, (iii) BR trimers and (iv) BR monomers. In all samples the first intermediate J is formed within 430±50 fs. No isotope effect is observed in the formation of J upon deuteration, in contrast to previous reports with much higher excitation energies. Thus proton movement to or from the retinal Schiff's base is not relevant during the first step. Comparing the data for trimeric and monomeric BR suggests an upper limit of 50 fs for the transfer of excitation energy from the excitonically coupled trimer to a single retinal chromophore

Energy transfer from retinal to amino acids — a time-resolved study of the ultraviolet emission of bacteriorhodopsin

Two-step excitation of retinal in bacteriorhodopsin by visible light is followed by an energy transfer to amino acids that is seen as fluorescent emission around 350 nm. The fluorescence spectrum obtained after two-step excitation (2 × 527 nm) differs from the fluorescence spectrum obtained after one-step ultraviolet excitation (263.5 nm) by a strongly quenched emission with a fluorescence lifetime of 10 ± 5 ps and a smaller spectral width. The two-step absorption process presumably selects tryptophan residues which strongly couple to the retinal chromophore

Early picosecond events in the photo cycle of Bacteriorhodopsin

The primary processes of the photochemical cycle of light-adapted bacteriorhodopsin (BR) were studied by various experimental techniques with a time resolution of 5 × 10-13 s. The following results were obtained. (a) After optical excitation the first excited singlet state S1 of bacteriorhodopsin is observed via its fluorescence and absorption properties. The population of the excited singlet state decays with a lifetime τ1 of ~0.7 ps (430 ± 50 fs) (52). (b) With the same time constant the first ground-state intermediate J builds up. Its absorption spectrum is red-shifted relative to the spectrum of BR by ~30 nm. (c) The second photoproduct K, which appears with a time constant of τ2 = 5 ps shows a red-shift of 20 nm, relative to the peak of BR. Its absorption remains constant for the observation time of 300 ps. (d) Upon suspending bacteriorhodopsin in D2O and deuterating the retinal Schiff base at its nitrogen (lysine 216), the same photoproducts J and K are observed. The relaxation time constants τ1 and τ2 remain unchanged upon deuteration within the experimental accuracy of 20%

Subpicosecond emission studies of bacterial reaction centers

The spontaneous emission of reaction centers from native and mutated Rhodobacter sphaeroides and from wild type Chloroflexus aurantiacus is investigated by fluorescence up-conversion with high temporal resolution. The time constant of 0.9 ps previously observed in transient absorption experiments on wild type reaction centers of Rhodobacter sphaeroides does not appear in the emission experiment. However, all investigated reaction centers display a biexponential decay of the emission with time constants in the 2 ps to 25 ps range. The experimental results are discussed within the frame of different reaction models including a possible sample heterogeneity or a transient electron transfer to the inactive pigment branch

Optical picosecond studies of bacteriorhodopsin containing a sterically fixed retinal

The photochemical behaviour of an analogous bacteriorhodopsin (9,12-Ph-BR) which contains the sterically fixed 9,12-phenylretinal has been investigated with picosecond spectroscopy. The following results have been obtained. No ground-state intermediate photoproduct is found in agreement with the previous observation that 9,12-Ph-BR does not exhibit proton pumping under illumination. The excited singlet state has a lifetime of τS = 10 ± 2 ps. This lifetime agrees favourably with the value calculated from the radiative lifetime τrad = 6.2 ns and the fluorescence quantum efficiency of 1.2·10−3. Excited-state absorption occurs which results in fluorescence in the ultraviolet region. These various observations differ drastically from the corresponding findings on bacteriorhodopsin. Most important for an understanding of the differences is the fact that 9,12-phenylretinal does not isomerize in the protein's binding site in contrast to retinal. Our data therefore suggest that the formation of the intermediate K observed in bacteriorhodopsin is accompanied by the all-trans to 13-cis isomerization

Observation of a bacteriochlorophyll anion radical during the primary charge separation in a reaction center

The primary light-induced charge separation in reaction centers of Rhodobacter sphaeroides was investigated with femtosecond time resolution. The absorption changes in the time range 100 fs to 1 ns observed after direct excitation of the primary donor P at 860 nm could only be explained by a kinetic model which uses three time constants. This finding supports the following reaction scheme: (i) the electronically excited primary donor P* decays with a time constant of 3.5 ps and populates a very short-lived intermediate involving a reduced accessory bacteriochlorophyll molecule; (ii) with a time constant of 0.9 ps the electron is transferred to the neighboring bacteriopheophytin molecule; and (iii) from there within 200 ps to the quinone