The pH dependence of the subpicosecond retinal photoisomerization process in bacteriorhodopsin: evidence for parallel photocycles.

The pH dependence of the subpicosecond decay of the retinal photoexcited state in bacteriorhodopsin (bR) is determined in the pH range 6.8-11.3. A rapid change in the decay rate of the retinal photoexcited state is observed in the pH range 9-10, the same pH range in which a rapid change in the M412 formation kinetics was observed. This observation supports the previously proposed heterogeneity model in which parallel photocycles contribute to the observed pH dependence of the M412 formation kinetics in bR.     

Transient absorption and photovoltage study of' self-assembled bacteriorhodopsin/polycation multilayer films

A series of organized (PDAC/PM)(n) (poly(diallyldimethylammonium chloride)/purple membrane) multilayer films were prepared by alternate adsorptions of positively charged PDAC polyelectrolyte and negatively charged purple membrane (PM). The kinetics of the photocycle of bacteriorhodopsin (bR) in PM was studied by flash photolysis and transient photovoltage methods. Although the orientation of the adsorbed bR depends on the pH of the PM suspension, the kinetics of the photo-induced reaction cycle in dehydrated films is independent of the deposition pH. In dry (PDAC/PM)(n) films the decay of the M intermediate to the initial bR state is multiexponential and delayed to several minutes for both orientations. A simultaneous two-exponential decay in millisecond time domain was observed at red wavelengths. The source of the red-shifted absorption is suggested to be the C(610) intermediate of the cis photocycle of bR.     

Proton transfer reactions in native and deionized bacteriorhodopsin upon delipidation and monomerization

We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is approximately 30%-40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is approximately 10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed.     

Oxygen activation catalyzed by methane monooxygenase hydroxylase component: proton delivery during the O-O bond cleavage steps

The effects of solvent pH and deuteration on the transient kinetics of the key intermediates of the dioxygen activation process catalyzed by the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been studied. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains a carboxylate- and oxygen-bridged binuclear iron cluster that catalyzes O2 activation and insertion chemistry. The diferrous MMOH-MMOB complex reacts with O2 to form a diferrous intermediate compound O (O) and subsequently a diferric intermediate compound P (P), presumed to be a peroxy adduct. The O decay reaction was found to be pH-independent within error at 4 degrees C (kobs = 22 +/- 2 s-1 at pH 7.7; kobs = 26 +/- 2 s-1 at pH 7.0). In contrast, the P formation rate was found to decrease sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kP = 9.1 +/- 0.9 s-1 achieved near pH 6.5. The formation of P was slower than the disappearance of O, indicating that at least one other undetected intermediate (P) must form in between. P decays spontaneously to the highly chromophoric intermediate, compound Q (Q). The decay rate of P matched the formation rate of Q, and both rates decreased sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kQ = 2.6 +/- 0.1 s-1 achieved near pH 6.5. No pH dependence was observed for the decay of Q. The formation and decay rates of P and the formation rate of Q decreased linearly with mole fraction of D2O in the reaction mixture. Kinetic solvent isotope effect values of kH/kD = 1.3 +/- 0.1 (P formation) and kH/kD = 1.4 +/- 0.1 (P decay and Q formation) were observed at 5 degrees C. The linearity of the proton inventory plots suggests that only a single proton is transferred in the transition state of the formation reaction for each intermediate. If these protons are transferred to the bound oxygen molecule, as formally required by the reaction stoichiometry, the data are consistent with a model in which water is formed concurrently with the formation of the reactive bis mu-oxo-binuclear Fe(IV) species, Q.     

Photocycle of dried acid purple form of bacteriorhodopsin.

The photocycle of dried bacteriorhodopsin, pretreated in a 0.3 M HCl solution, was studied. Some properties of this dried sample resemble that of the acid purple suspension: the retinal conformation is mostly all-trans, 15-anti form, the spectrum of the sample is blue-shifted by 5 nm to 560 nm, and it has a truncated photocycle. After photoexcitation, a K-like red-shifted intermediate appears, which decays to the ground state through several intermediates with spectra between the K and the ground state. There are no other bacteriorhodopsin-like intermediates (L, M, N, O) present in the photocycle. The K to K' transition proceeds with an enthalpy decrease, whereas during all the following steps, the entropic energy of the system decreases. The electric response signal of the oriented sample has only negative components, which relaxes to zero. These suggest that the steps after intermediate K represent a relaxation process, during which the absorbed energy is dissipated and the protein returns to its original ground state. The initial charge separation on the retinal is followed by limited charge rearrangements in the protein, and later, all these relax. The decay times of the intermediates are strongly influenced by the humidity of the sample. Double-flash experiments proved that all the intermediates are directly driven back to the ground state. The study of the dried acid purple samples could help in understanding the fast primary processes of the protein function. It may also have importance in technical applications.     

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.     

Effects of pressure and temperature on the M412 intermediate of the bacteriorhodopsin photocycle. Implications for the phase transition of the purple membrane

The effects of pressure and temperature on the decay kinetics of the M412 (M) intermediate in the photocycle of bacteriorhodopsin were studied to provide information about the phase transitions of the purple membrane lipids. The activation volume (delta V++) for the decay of M is expected to be different below and above a phase transition. However, no abrupt change in delta V++ was found from 3.5 degrees to 60 degrees C. But a sharp break was observed in a plot of the logarithm of the rate of M decay vs. pressure. Extrapolation of this break point to standard atmospheric pressure gives a temperature of -42 degrees C, which probably corresponds to the phase transition of the purple membrane lipids. This conclusion is supported by studies of the effect of pressure on the M kinetics of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine vesicles, whose phase transition has previously been characterized.     

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.     

Photocyclization of a conformationally constrained 2-vinylbiphenyl.

The synthesis and photochemical behavior of a rigid analog of syn-2-vinylbiphenyl are reported. This analog undergoes quantitative conversion to a tetrahydrobenzanthracene derivative upon irradiation in fluid solution. Product formation is proposed to occur via photocyclization to yield an unstable intermediate followed by an intramolecular thermal hydrogen shift. The intermediate can be observed upon irradiation at low temperature either in a rigid glass or in liquid propane solution. The temperature dependence of its decay is indicative of a tunneling mechanism for the hydrogen shift process.     

The ESR determination of protein alpha-helicity.

Experimental data are summarized on decay kinetics of uv-induced paramagnetic centers in globular proteins. The PC decay rate in the absence of oxygen in α helical regions is shown to differ always by about two orders from that in nonhelical regions of the polypeptide chain. This effect can be utilized to estimate the degree of α helicity in the dry state, in solution, and in the frozen solution of proteins.     

Denitrification by a mixture of bacterial strains derived from an upflow sludge blanket reactor, following entrapment in sol-gel glass

Whole cell extracts of a bacterial mixture grown in an upflow sludge blanket reactor used for drinking water denitrification, were successfully entrapped into sol-gel glass, and afterwards air dried. The sol-gel complex was able to reduce sodium nitrate and nitrite solutions. Compared with free whole cell extract, the sol-gel complex required twice as much time for nitrate and nitrite reduction. This study is the first successful attempt to entrap denitrifier reductases for experimental denitrification into sol-gel glass. Denitrifying enzymes entrapped into sol-gel glass matrix, can be used several times without significant loss in specificity, but gradual loss in reactivity occurs, probably due to electron donor depletion.     

Temperature dependence of tryptophan fluorescence lifetime in aqueous glycerol and trehalose solutions

The temperature dependences of tryptophan fluorescence decay kinetics in aqueous glycerol and 1 M trehalose solutions were examined. The fluorescence decay kinetics were recorded in the spectral region of 292.5–417.5 nm with nanosecond time resolution. The kinetics curves were approximated by the sum of three exponential terms, and the spectral distribution (DAS) of these components was determined. An antisymbatic course of fluorescence decay times of two (fast and medium) components in the temperature range from –60 to +10°C was observed. The third (slow) component showed only slight temperature dependence. The antisymbatic behavior of fluorescence lifetimes of the fast and medium components was explained on the assumption that some of the excited tryptophan molecules are transferred from a short-wave-length B-form with short fluorescence lifetime to a long-wavelength R-form with an intermediate fluorescence lifetime. This transfer occurred in the indicated temperature range.     

Time-Resolved FTIR Studies of Sensory Rhodopsin II (NpSRII) from Natronobacterium pharaonis: Implications for Proton Transport and Receptor Activation

The photocycle of the photophobic receptor from Natronobacterium pharaonis, NpSRII, is studied by static and time-resolved step-scan Fourier transform infrared spectroscopy. Both low-temperature static and time-resolved spectra resolve a K-like intermediate, and the corresponding spectra show little difference within the noise of the time-resolved data. As compared to intermediate K of bacteriorhodopsin, relatively large amide I bands indicate correspondingly larger distortions of the protein backbone. The time-resolved spectra identify an intermediate L-like state with surprisingly small additional molecular alterations. With the formation of intermediate M, the Schiff-base proton is transferred to the counterion Asp-75. This state is characterized by larger amide bands indicating larger distortions of the protein. We can identify a second M state that differs only in small-protein bands. Reisomerization of the chromophore to all-trans occurs with the formation of intermediate O. The accelerated decay of intermediate M caused by azide results in another red-shifted intermediate with a protonated Schiff base. The chromophore in this state, however, still has 13-cis geometry. Nevertheless, the reisomerization is still as slow as under the conditions without azide. The results are discussed with respect to mechanisms of the observed proton pumping and the possible roles of the intermediates in receptor activation.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

Dynamics of Re(2,2'-bipyridine)(CO)3Cl MLCT formation and decay after picosecond pulsed X-ray excitation and femtosecond UV excitation.

The dynamics of Re(2,2'-bipyridine)(CO)3Cl MLCT state formation and decay were determined after femtosecond UV laser excitation and picosecond pulsed X-ray excitation, in an N,N-dimethylformamide (DMF) solution as well as in its solid form. At room temperature, after UV excitation, this MLCT excited state emits both in DMF solution and in the solid form. Transient absorption spectra were measured in solution at various delay times following excitation by a 160 fs, 390 nm laser pulse. There was a prompt absorption increase at around 460 nm occurring within the pump probe convolution (<1 ps), which was assigned to the formation of the 3MLCT state. This transient absorbance was constant over 100 ps. In contrast to the solution state, in the solid state, the emission maximum slightly red-shifts with increasing time after laser excitation. In both solid and solution the emission rises within the system response time. The solid sample exhibited a 1.4 ns emission decay that was not observed for the solution sample. The emission rise from a solid sample after 20 ps pulsed X-ray excitation was significantly slower than the system's time resolution. It is proposed that kinetically energetic electrons are ejected following X-ray induced ionisation, creating ionised tracks in which energetic cations and electrons take time to recombine yielding delayed 3MLCT states that emit.     

Rate constants for the first two chemical steps of eumelanogenesis

The kinetics of the initial cyclization and redox exchange reactions involved in the eumelanogenic pathway have been studied previously but because of the difficulty of detecting the intermediate cyclodopa by optical means (because its absorbance is in the same range as dopa which is present in excess in the experimental system) no accurate value for the redox exchange reaction has so far been obtained and there is no available analytical methodology that can be applied to the successive first- and second-order reactions involved. We have synthesized cyclodopa and examined the kinetics of the formation of dopachrome following the pulse radiolytic generation of dopaquinone in its presence. From this direct measurement we determined that the rate constant of the reaction between cyclodopa and dopaquinone is 5.3 x 10(6)/M/s. Employing this value in a computational model of the combined cyclization and redox exchange reactions we calculate that the observed kinetics of dopaquinone decay and dopachrome formation are compatible with a cyclization rate constant of 3.8/s.     

Intermediates in the refolding of ribonuclease at subzero temperatures. 3. Multiple folding pathways

The kinetics of refolding of ribonuclease A have been measured at -15 degrees C by monitoring the intrinsic fluorescence and absorbance signals from the six tyrosine residues. For each probe multiphasic kinetics were observed. The burial of tyrosine residues, as determined by the change in absorbance at 286 nm, revealed four phases, whereas the kinetics of refolding monitored by fluorescence revealed only two phases. The rates of the transients detected by fluorescence were independent of pH. One of the faster transients detected by delta A286 involved a decrease in absorbance, which is consistent with solvent exposure, rather than burial, and suggests the possibility of an abortive partially folded intermediate in the earlier stages of folding. Double-jump unfolding assays were used to follow the buildup and decay of an intermediate in the refolding reaction at -15 degrees C. At both pH* 3.0 and pH* 6.0 the maximum concentration of the intermediate was 25-30% of the total protein. The existence of a second pathway of slow folding was inferred from the difference in rate of formation of native enzyme and breakdown of the observed intermediate, and by computer simulations. In addition, the unfolding assay demonstrated that 20% of the unfolded protein was converted to native at a much faster rate, consistent with observations in aqueous solution that 80% of unfolded ribonuclease A consists of slow-folding species. Kinetics and amplitude data from these and other refolding experiments with different probes were used to develop possible models for the pathway of refolding. The simplest system consistent with the results for the slow-refolding species involves two parallel pathways with multiple intermediates on each of them. Several independent lines of evidence indicate that about 30% of the unfolded state refolds by the minor pathway, in which the slowest observed phase is attributed to the isomerization of Pro-93. The major pathway involves 50% of the unfolded state; the reason why it refolds slowly is not apparent. A native-like intermediate is formed considerably more rapidly in the major slow-refolding pathway, compared to the minor pathway.     

Acid denaturation and refolding of green fluorescent protein

Green fluorescent protein from the jellyfish Aequorea victoria can serve as a good model protein to understand protein folding in a complex environment with molecular chaperones and other macromolecules such as those in biological cells, but little is known about the detailed mechanisms of the in vitro folding of green fluorescent protein itself. We therefore investigated the kinetic refolding of a mutant (F99S/M153T/V163A) of green fluorescent protein, which is known to mature more efficiently than the wild-type protein, from the acid-denatured state; refolding was observed by chromophore fluorescence, tryptophan fluorescence, and far-UV CD, using a stopped-flow technique. In this study, we demonstrated that the kinetics of the refolding of the mutant have at least five kinetic phases and involve nonspecific collapse within the dead time of a stopped-flow apparatus and the subsequent formation of an on-pathway intermediate with the characteristics of the molten globule state. We also demonstrated that the slowest phase and a major portion of the second slowest phase were rate-limited by slow prolyl isomerization in the intermediate state, and this rate limitation accounts for a major portion of the observed kinetics in the folding of green fluorescent protein.     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

Pathways of the rise and decay of the M photointermediate(s) of bacteriorhodopsin

The photocycle of bacteriorhodopsin (BR) was studied at alkaline pH with a gated multichannel analyzer, in order to understand the origins of kinetic complexities in the rise and decay of the M intermediate. The results indicate that the biphasic rise and decay kinetics are unrelated to a photoreaction of the N intermediate of the BR photocycle, proposed earlier by others [Kouyama et al. (1988) Biochemistry 27, 5855-5863]. Rather, under conditions where N did not accumulate in appreciable amounts (high pH, low salt concentration), they were accounted for by conventional kinetic schemes. These contained reversible interconversions, either M in equilibrium with N in one of two parallel photocycles or L in equilibrium with as well as M in equilibrium with N in a single photocycle. Monomeric BR also showed these kinetic complications. Conditions were then created where N accumulated in a photo steady state (high pH, high salt concentration, background illumination). The apparent increase in the proportion of the slow M decay component by the background illumination could be quantitatively accounted for with the single photocycle model, by the mixing of the relaxation of the background light induced photo steady state with the inherent kinetics of the photocycle. Postulating a new M intermediate which is produced by the photoreaction of N was neither necessary nor warranted by the data. The difference spectra suggested instead that absorption of light by N generates only one intermediate, observable between 100 ns and 1 ms, which absorbs near 610 nm. Thus, the photoreaction of N resembles in some respects that of BR containing 13-cis-retinal.