The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin: mechanism and evidence for two M species

The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin is investigated both for the native pigment and its D96N mutant. The experimental setup is based on creating the M intermediate by a first pulse, followed by a (blue) laser pulse which drives the back photoreaction of M. Experiments are carried out varying the delay between the two pulses, as well as the temperature over the -25 degrees C-20 degrees C range. It is found that the kinetic patterns of the M back photoreaction change with time after the generation of this intermediate. The data provide independent evidence for the suggestion of a photocycle mechanism based on two distinct M intermediates. They are thus in keeping with the consecutive model of Varo and Lanyi (Biochemistry 30, 5016-5022; 1991), although they cannot exclude other models such as those based on branched or parallel cycles. More generally, we offer a "photochemical" approach to discriminating between intermediate stages in the photocycle which does not depend on spectroscopic and/or kinetic data. While markedly affecting the rate of the M --> N transition in the photocycle, the rate of the thermal step in back photoreaction of M, at both room and low temperatures, is not significantly affected by the D96N mutation. It is proposed that while Asp 96 is the Schiff-base protonating moiety in the M --> N transition, another residue (most probably Asp 85) reprotonates the Schiff base following light absorption by M.     

A priori resolution of the intermediate spectra in the bacteriorhodopsin photocycle: the time evolution of the L spectrum revealed

Resolution of the spectra of the intermediates in the photocycle of wild-type bacteriorhodopsin (BR) was achieved by singular value decomposition with exponential-fit-assisted self-modeling (SVD-EFASM) treatment of multichannel difference spectra measured at 5 degrees C during the course of the photocycle. New is the finding that two spectrally distinct L intermediates, L(1) and L(2), form sequentially. Our conclusion is that the photocycle is more complex than most published schemes. The dissection of the spectrally different L forms eliminates stoichiometric discrepancies usually appearing as systematically varying total intermediate concentrations before the onset of BR recovery. In addition, our analysis reveals that the red tails in the spectra of K and L(1) are more substantial than those of L(2) and BR. We suggest that these subtle differences in the shapes of the spectra reflect torsional and/or environmental differences in the retinyl chromophore.     

锡负载量对PtSn/Al2O3催化丙烷脱氢性能的影响

采用羰基合成-浸渍法制备了不同Pt/Sn摩尔比(3:1, 1:1, 1:2和1:3)的PtSn/Al₂O₃催化剂, 利用N₂吸附-脱附实验、 X射线衍射(XRD)、 透射电子显微镜(TEM)、 吡啶吸附红外光谱(Py-IR)和热重-差热分析(TG-DTA)等手段对其进行了表征, 研究了Sn负载量对PtSn/Al₂O₃的结构性质及催化丙烷脱氢性能的影响. 结果表明, 制备的PtSn/Al₂O₃具有较高的丙烯选择性和稳定性. 当Pt/Sn摩尔比为3:1和1:1时, 铂和锡在催化剂上主要以Pt₃Sn和PtSn合金形式存在, 合金的形成明显改善了催化剂的脱氢性能, 可抑制金属颗粒的高温烧结; 当Pt/Sn摩尔比为1:2和1:3时, 铂主要以金属形式存在. 随着Sn负载量的增加, 催化剂上L酸性位逐渐减少, 丙烷转化率降低, 丙烯选择性增加, 同时促使反应积炭从金属表面向载体迁移, 改善了催化剂的稳定性.     

Thermal Equilibrium Between Radiation and Matter

In 1916, Einstein rederived the blackbody radiation law of Planck that originated the idea of quantized energy one hundred years ago. For this purpose, Einstein introduced the concept of transition probability, which had a profound influence on the development of quantum theory. In this article, we adopt Einstein's assumptions with two exceptions and seek the statistical condition for the thermal equilibrium of matter without referring to the inner details of either statistical thermodynamics or quantum theory. It is shown that the conditions of thermodynamic equilibrium of electromagnetic radiation and the energy balance of thermal radiation by the matter, between any of its two energy-states, not only result in Planck's radiation law and the Bohr frequency condition, but they remarkably yield the law of the statistical thermal equilibrium of matter: the Maxwell–Boltzmann distribution. Since the transition probabilities of the modern quantum theory of radiation coincide with their definition in Einstein's theory of blackbody radiation, the presented deduction of the Maxwell–Boltzmann distribution is equally valid within the bounds of modern quantum theory. Consequently, within the framework of the fundamental assumptions, the Maxwell–Boltzmann distribution of energy-states is not only a sufficient, but a necessary condition for thermal equilibrium between the matter and radiation.     

X-Ray Diffraction Studies of Bacteriorhodopsin. Determination of the Positions of Mercury Label at Several Engineered Cysteine Residues

Abstract— The single cysteine-containing bacteriorhodopsin mutants F27C, L100C, T170C, F171C and I222C were labeled with p -chloromercuribenzoic acid, which specifically reacts with sulfhydryl groups. These cysteines should be located at the cytoplasmic ends of the transmembrane helices A, C, F or G. We determined the positions of the bound mercury atoms by X-ray diffraction of purple membrane films, with better than 1 Å accuracy. The determined mercury positions were compared with the structural model from cryoelectron microscopy (N. Grigorieff, T. A. Ceska, K. H. Downing, J. M. Baldwin and R. Henderson, J. Mol. Biol 259, 393-421, 1996). Given that the distance between the mercury and the Cα atom of the cysteine in the xy plane must be shorter than 4.5 Å and that the mercury atom is located at the δ position, the positions obtained for the mercury labels agree with their expected positions from the structural model. The present results give a rationale for detecting structural changes upon illumination as shifts occur in the mercury label position.     

Infrared Monitoring of Interlayer Water in Stacks of Purple Membranes†

The thermodynamic behavior of films of hydrated purple membranes from Halobacterium salinarum and the water confined in it was studied by Fourier transform infrared spectroscopy in the 180–280 K range. Unlike bulk water, water in the thin layers sandwiched between the biological membranes does not freeze at 273 K but will be supercooled to ～256 K. The melting point is unaffected, leading to hysteresis between 250 and 273 K. In its heating branch, a gradually increasing light‐scattering by ice is observed with rate‐limiting kinetics of tens of minutes. Infrared (IR) spectra decomposition provided extinction coefficients for the confined water vibrational bands and their changes upon freezing. Because of the hysteresis, at any given temperature in the 255–270 K range, the interbilayer water could be either liquid or frozen, depending on thermal history. We find that this difference affects the dynamics of the bacteriorhodopsin photocycle in the hysteresis range: the decay of the M and N states and the redistribution between them are different depending on whether or not the water was initially precooled to below the freezing point. However, freezing of interbilayer water does block the M to N transition. Unlike the water, the purple membrane lipids do not undergo any IR‐detectable phase transition in the 180–280 K range.     

pH-dependent transitions in xanthorhodopsin

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.