Photochemical Cycle Of Bacteriorhodopsin Research Articles

Volume and Enthalpy Changes of Proton Transfers in the Bacteriorhodopsin Photocycle Studied by Millisecond Time-Resolved Photopressure Measurements

The volume and enthalpy changes associated with proton translocation steps during the bacteriorhodopsin (BR) photocycle were determined by time-resolved photopressure measurements. The data at 25 degrees C show a prompt increase in volume followed by two further increases and one decrease to the original state to complete the cycle. These volume changes are decomposed into enthalpy and inherent volume changes. The positive enthalpy changes support the argument for inherent entropy-driven late steps in the BR photocycle [Ort, D. R., and Parson, W. M. (1979) Enthalpy changes during the photochemical cycle of bacteriorhodopsin. Biophys. J. 25, 355-364]. The volume change data can be interpreted by the electrostriction effect as charges are canceled and formed during the proton transfers. A simple glutamic acid-glutamate ion model or a diglutamate-arginine-protonated water charge-delocalized model for the proton-release complex (PRC) fit the data. A conformational change with a large positive volume change is required in the slower rise (M --> N of the optical cycle) step and is reversed in the decay (N --> O --> BR) steps. The large variation in the published values for both the volume and enthalpy changes is greatly ameliorated if the values are presented per absorbed photon instead of per mole of BR. Thus, it is the highly differing assumptions about the quantum or reaction yields that cause the variations in the published results.

On the action spectrum of the photoelectric transients of bacteriorhodopsin in solid-state films

We have measured the time-resolved charge displacement currents of bacteriorhodopsin (BR) from Halobacterium halobium in multilayer arrays of well-oriented purple membranes in photoelectric cells with the sandwich electrode configuration: SnO 2 |BR|Ag. The purple membrane cells were irradiated by dye laser pulses entering the cells through the light-transmitting SnO 2 electrode. The photochemical cycle of BR was found to be reversible in these solid films, and we observed a major decay component for the M intermediate with a decay time of 46 ± 3 ms as measured by the transient optical absorption at 412 nm. We also observed a minor component (≤5% of the total amplitude) with a decay time of 0.97 ± 0.15 s. We measured the early action spectrum for the instrument limited amplitudes of the photoelectric behaviour: this action spectrum differed from the absorption spectrum of the absorbing BR pigment. However, the action spectrum measured 500 μs after flash excitation was in agreement with the absorption spectrum of BR. We attribute the nature of the early action spectrum to the presence of red and blue absorbing species which are not capable of pumping protons across the purple membrane bilayer. We also measured the charge displacement currents when the purple membrane cells were perturbed by small applied voltages with a specific polarity: for conduction of negative charges in the same direction as vectorial proton transport, enhanced transient photocurrents resulted. We interpret these enhanced photocurrents in terms of an enhancement of observed charge separation resulting from a reduced back reaction from the K intermediate of the photocycle.

Photovoltaic properties of purple membrane Langmuir-Blodgett films

Langmuir films of pure purple membranes with no added lipids were deposited on solid substrates by the horizontal transfer method. The photovoltaic properties of purple membrane Langmuir-Blodgett (LB) films were studied in correlation with the photochemical cycle of bacteriorhodopsin. The morphology of the air-water interface film transferred onto a chip of silicon wafer is briefly described as a characterization for purple membrane LB films.

BLEACHING OF BACTERIORHODOPSIN IN PURPLE MEMBRANE, BROWN HOLO‐MEMBRANE ANDL–1690‐SOLUBILIZED MONOMERS WITH HYDROXYLAMINE IN LIGHT AND THE DARK

Abstract— The reactions of hydroxylamine with bacteriorhodopsin in the states of the purple membrane, the brown holo‐membrane andL–1690‐solubilized monomers were studied in light and the dark. The bleaching rate was strongly dependent on the state of the bacteriorhodopsin and largely enhanced by light. Analysis of the isomeric composition of the resultant retinaloximes by high performance liquid chromatography (HPLC) showed that more all‐trans retinaloximes were present in the dark than 13‐cis retinaloximes. However, under illumination, the relative amounts of 13‐cis retinaloximes increased greatly, indicating the occurrence of trans to cis isomerization during the photochemical cycle of bacteriorhodopsin. Molar extinction coefficients of 13‐cis retinaloximes were determined to estimate the isomeric composition of retinaloximes by HPLC analysis.

The photochemical cycle of bacteriorhodopsin has no refractory period

Double-pulse experiments were used to probe the existence of a refractory period in the photochemical cycle of bacteriorhodopsin. We demonstrate using three different approaches that no refractory period exists in purple membranes, cell vesicles or intact cells. First, we showed by applying different time delays between two flashes that every molecule which regained the initial optical state could immediately recycle again. Second, we found no difference in the effectiveness of the first and second flash when the intensity was varied. Third, different steady-state levels of the intermediates were produced with different intensities of continuous background illumination. No difference in effectivity of the first and second flash could be detected.

Time-resolved protein fluorescence studies of intermediates in the photochemical cycle of bacteriorhodopsin.

The photolysis-induced changes in the protein fluorescence intensity (at 320 nm) during the proton-pumping cycle of bacteriorhodopsin were examined by a delayed two-pulse technique in the time range 1 microsecond-20 msec at room temperature. No detectable change in the protein fluorescence intensity was observed on the earliest time scale within the lifetime of the intermediate K590, when retinal apparently undergoes the largest structural changes. The time dependence of the relative changes in fluorescence intensity did, however, display a close correlation with the population of the L550 and M412 intermediates. From a computer numerical fit of the data, with available published kinetic parameters, the protein fluorescence quantum yields of the K590, L550, and M412 intermediates are found to be 1.0, 0.92, and 0.80 of that for native bR570, respectively. The probable mechanisms of the observed fluorescence quenching during the photochemical cycle are qualitatively discussed.

Evidence for a model of regeneration of a protonated species, bR, from a phototransient, M, in the photochemical cycle of bacteriorhodopsin from Halobacterium halobium [proceedings

Evidence for a model of regeneration of a protonated species, bR, from a phototransient, M, in the photochemical cycle of bacteriorhodopsin from Halobacterium halobium [proceedings

Bacteriorhodopsin in liposomes. I. A description using irreversible thermodynamics

A comprehensive description of light-induced ion transport in bacteriorhodopsin liposomes is presented. Linear irreversible thermodynamics and the chemiosmotic theory serve as theoretical bases for the formulation of a limited number of fundamental equations. In these equations mechanistic parameters characterize the dependence of ion movement and flux through the photochemical cycle of bacteriorhodopsin on electrochemical potential differences and a so-called light affinity. By making appropriate steady-state assumptions and carrying out mathematical reduction experimentally testable expressions, still containing the mechanistic parameters, are obtained. In the accompanying article rigid trials to falsify these expressions are shown to be unsuccessful.

Enthalpy changes during the photochemical cycle of bacteriorhodopsin

We have used a capacitor microphone calorimeter to measure rapid enthalpy changes that occur when bacteriorhodopsin-containing membrane fragments are excited with short flashes of light. We resolved the enthalpy changes into three phases. At about 100 microsecond after the flash, the bacteriorhodopsins converted into metastable states have an enthalpy about 15-20 kcal mol-1 greater than the enthalpy before excitation. Some of this energy (approximately 10 kcal) is then released to the surroundings as the membrane fragments release protons to the solution. After proton release and before proton rebinding, a large amount of heat is released to the surroundings, equivalent to about 40-45 kcal/mol of bacteriorhodopsin reacting. At this point the energy of the system is about 35 kcal/mol less than it was before the flash; i.e., the system has released all of the energy of the photon (49 kcal/E) plus an additional 35 kcal/mol. Nevertheless, the free energy of the system must still be greater than it was originally, because relaxation to the original state occurs spontaneously. An entropy decrease of at least 125 cal/mol per deg is required to compensate for the heat release. An entropy decrease of this magnitude implies a major increase in molecular order in the purple membrane.

Photoelectric activity of dried, oriented layers of purple membrane from halobacterium halobium

Summary Dried layers of purple membranes containing bacteriorhodopsin oriented by an electric field during drying were investigated by photoelectric and spectroscopic methods. There is evidence to show that the photochemical cycle of bacteriorhodopsin in a l&w hydration state is intact not only spectroscopically but also electronically in these layers. It has been observed that due to the blue-light excitation of the bleached form an intermediate absorbing at 520 nm is enriched. Thus this form seems to occur in the inhibitory pathway of bacteriorhodopsin.

Primary intermediates in the photochemical cycle of bacteriorhodopsin

Picosecond studies of the primary photochemical events in the light-adapted bacteriorhodopsin, bR570, indicate that the first metastable intermediate K610 is formed with a rise time of 11 ps. Difference spectra obtained at 50 ps after excitation show that K610 is the same species as that trapped in low temperature glasses. A precursor species (S) of the K610 intermediate has been observed which is red shifted with respect to K610 and is formed within the 6-ps time width of the excitation pulse. The formation of the precursor has no observable thermal dependence between 298 degrees and 1.8 degrees K. The formation of K610 has a very low thermal barrier and at very low temperatures, the rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation of K610 has a moderate deuterium isotope effect of kH/kD approximately 1.6 at 298 degrees K and 2.4 at 4 degrees K. The mechanism for formation of K610 is found to involve a rate-limiting proton transfer which occurs by tunneling at low temperatures.

Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

A simple technique is described that uses a continuous wave laser with electromechanical modulation to obtain time-resolved Raman spectra of transient species on the millisecond timescale. The time behavior of the vibrational bands of the intermediates involved in the proton pumping of bacteriorhodopsin is determined. From these results, along with resonance enhancement and power dependence studies, the bands that appear in the continuous wave Raman spectrum of bacteriorhodopsin can be assigned to three intermediates in the photochemical cycle of bacteriorhodopsin, bR570, bL550, and bM412. The Raman spectra of bR570 and bM412 are compared with published spectra of model Schiff bases of all-trans and 13-cis retinal.

Flash photometric experiments on the photochemical cycle of bacteriorhodopsin.

The photochemical reaction cycle of bacteriorhodopsin was investigated by means of flash photometric methods. Three different intermediates with absorption maxima at about 630 nm, 411 nm, and 646 nm could be detected. Kinetic data of the occurrence of these intermediates were obtained from isolated purple membrane in different mediums and from intact halobacteria. An activation energy of 14.1 +/- 0.4 kcal-mol-1 and of about 19 kcal-mol-1 for formation of bacteriorhodopsin 411 and of bacteriorhodopsin 565, resp., was calculated. pH-changes in the medium caused by the reaction cycle of bacteriorhodopsin were detected by use of the pH-indicator bromocresol green.