Allosteric intermediates in hemoglobin. 1. Nanosecond time-resolved circular dichroism spectroscopy.

Time-resolved circular dichroism (TRCD) studies performed on photolyzed hemoglobin-CO complex (HbCO) probe room temperature protein relaxations in Hb, including the R --> T allosteric transition. TRCD spectroscopy of photolysis intermediates in the near-UV (250-400 nm) spectral region provides a diagnostic for T-like structure at the alpha 1 beta 2 interface via the effect of quaternary structure on the UV CD of aromatic residues. The TRCD of porphyrin-based transitions in the UV and Soret regions, reflecting transition-dipole couplings between hemes and aromatic residues over a radius wide enough to permit heme-interface and inter-dimer interactions, is modulated by the tertiary and quaternary structure of photolysis intermediates. In the allosteric core model of Hb cooperativity, Fe-CO bond breakage initiates a heme structural change, thought to be heme doming, that is transmitted to the alpha 1 beta 2 interface via the F helix. The TRCD results, analyzed in light of kinetic information from time-resolved absorption studies, suggest specific features for the mechanism by which the ensuing tertiary and quaternary conformational changes propagate through the protein. In particular, the UV-TRCD indicates that the alpha 1 beta 2 interface responds within several hundred nanoseconds to initial events at the heme by shifting from an R toward a T-like interface. The appearance of T-like character at the alpha 1 beta 2 interface tens of microseconds before the appearance of equilibrated T state deoxyHb indicates that the R --> T transition in photolyzed HbCO is a stepwise process, as previously suggested by time-resolved resonance Raman studies.

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 detergent environments on the photocycle of purified monomeric bacteriorhodopsin.

Time-resolved difference spectra have been obtained for the photocycle of delipidated bacteriorhodopsin monomers (d-BR) in six different detergent micelle environments that were prepared by two new detergent-exchange techniques. A global kinetic analysis of the photocycle spectra for d-BR in each detergent environment was performed. Comparison of these results with those obtained for the photocycle of bacteriorhodopsin in purple membrane (PM) shows that there is one fewer kinetically distinguishable process for monomeric BR between the decay of the K intermediate and the rise of the M intermediate. Assuming a sequential pathway occurs in the photocycle, it appears that the equilibrium between the L and M intermediates is reached much more rapidly in the detergent micelles. This is attributed to a more direct interaction between Asp-85 and the proton on the nitrogen of the Schiff base of retinal for BR in the detergents. Equilibrium concentrations of late photocycle intermediates are also altered in detergents. The later steps of the photocycle, including the decay of the M intermediate, are slowed in detergents with rings in their hydrocarbon region. This is attributed to effects on conformational changes occurring during the decay of M and/or other later photocycle intermediates. The lifetime of dark adaptation of light-adapted d-BR in different detergent environments increases in environments where the lifetime of the M intermediate increases. These results suggest that the high percentage of either unsaturated or methyl-branched lipids in PM and the membranes of other retinal proteins may be important for their effective functioning.

Correlation between absorption maxima and thermal isomerization rates in bacteriorhodopsin.

The reported rates of thermal 13-cis to all-trans isomerization of the protonated Schiff base of retinal (PSBR) in solution and in bacteriorhodopsin (BR) are shown to be correlated with the red shift in the absorption maximum of the chromophore, though the linear fit is different for BR and for a model PSBR in solution. Because the red shift in the absorption has been previously shown to be correlated with pi-electron delocalization in the chromophore, this suggests that the thermal isomerization rate is largely regulated by the amount of double bond character in the chromophore. Because the linear fit of isomerization rates with absorption maxima is different for BR and the model PSBR, specific interactions of the protein with the chromophore must also be a factor in determining thermal isomerization rates in BR. A model of the later steps in the photocycle of BR is presented in which the 13-cis to all-trans thermal isomerization occurs during the O intermediate.

Time-resolved absorption, circular dichroism, and emission of tRNA. Evidence that the photo-cross-linking of 4-thiouridine in tRNA occurs from the triplet state.

The time-resolved optical density (TROD) and time-resolved circular dichroism (TRCD) spectra of the lowest triplet state of 4-thiouridine (4t-Urd) in aqueous solutions of tRNA are reported. The TROD spectrum is consistent with the triplet state being primarily in the thione tautomer. The intersystem crossing yield to the triplet is 0.35 and 0.27 (+/- 10%), respectively, with and without 10(-2) Mg2+ added to the solution. Upon addition of increasing amounts of I- to solutions of tRNA, the initial triplet yield decreases, the rate of the observed triplet decay increases, and the quantum yield of internal photo-cross-linking decreases for the 4t-Urd chromophore. The results show quantitatively that the near-UV-induced photo-cross-linking reaction in tRNA occurs from the triplet state of 4t-Urd. From the TRCD spectrum the dissymmetry factor (delta epsilon/epsilon) of some of the triplet-triplet absorption bands is shown to be significantly larger than for any of the ground-state absorption bands. Two CD transitions are seen in the triplet-triplet spectrum which are obscured in the TROD spectrum by the strong ground-state bleaching signal near 335 nm. This shows that TRCD may be useful, in some cases, in locating electronic transitions that are not observed in TROD spectra.

Time-resolved circular dichroism and absorption studies of the photolysis reaction of (carbonmonoxy)myoglobin.

Time-resolved circular dichroism (TRCD) and absorption spectroscopy are used to follow the photolysis reaction of (carbonmonoxy)myoglobin (MbCO). Following the spectral changes associated with the initial loss of CO, a subtle change is observed in the visible absorption spectrum of the Mb product on a time scale of a few hundred nanoseconds. No changes are seen in the CD spectrum of Mb in the visible and near-UV regions subsequent to the loss of CO. The data suggest the existence of an intermediate found after ligand loss from MbCO that is similar in structure to the final Mb product.

A time-resolved spectral study of the K and KL intermediates of bacteriorhodopsin.

Nanosecond time-resolved absorption measurements on the photolysis products of bacteriorhodopsin (BR) in intact membranes are reported. At room temperature in fluid solution a single intermediate (KL) is seen 10 ns after excitation. Both spectral and kinetic results are consistent with the KL intermediate converting to the L intermediate by a single first order reaction. The observed temperature-dependent rate has the Arrhenius parameters: Ea = 10.5 kcal/mol, A = 5 x 10(13) s-1. The precursor to the KL intermediate is also observed. Its spectral character is consistent with the K intermediate which has been previously reported. The current data is consistent with a linear sequence in the BR photocycle for K, KL, and L in room temperature fluid solution. Differences in the spectral characteristics of the K intermediates described here and elsewhere are discussed in terms of differences in the microenvironment around the retinal moiety and the affect this may have on the conformation of the chromophore.

A new approach to understanding the initial step in visual transduction.

Data from picosecond spectroscopic studies of the formation kinetics of bathorhodopsin upon photolysis of rhodopsin and isorhodopsin was analyzed in terms of the Englman-Jortner theory of radiationless transitions. It was found that low frequency vibrations of the protein and/or chromophore are important in coupling bathorhodopsin to its precursor. The results were consistent with a mechanism for bathorhodopsin formation involving only a simple chromophore isomerization. A similar analysis of the formation kinetics of the K state of bacteriorhodopsin showed that different low frequency vibrations than those calculated for rhodopsin couple it to its precursor. The frequency of these vibrations increases upon deuteration for rhodopsin, while it decreases upon deuteration for bacteriorhodopsin. This points out the importance the specific protein matrix has on the primary photolysis reaction.