The rate constants of the nonradiative decay process of the excited singlet state of trans-stilbene and transl,l'-biindanylidene ("stiff" stilbene) were measured in liquid n-alkanes at room temperature. These data were compared with the two models for isomerization in condensed phases proposed by Kramers and by Grote and Hynes. For trans-stilbene, the agreement with Kramers' model was unsatisfactory, whereas better agreement was obtained with Grote and Hynes' model. The data for stiff stilbene agreed well with Kramers' model in the Smoluchowski limit. These results were explained in terms of the shape of the potential energy barrier which separates the trans from the twisted configuration of the excited singlet state of these molecules. It is suggested that the deviation between theory and experiment is a result of a qualitative difference in the microscopic solvent-solute collisional interaction when the solvent molecules are longer than stilbene. The best fit of theory to experiment for trans-stilbene requires the use of an unphysically low (8 cm -1) frequency for the top of the barrier to isomerization.
The fluorescence decay characteristics of Mb, MbCO, metMb (sperm whale), metMb (yellowfin tuna), and their apo derivatives were determined by using a picosecond streak camera and time-correlated single photon counting. The emission is dominated by tryptophans that transfer their energy to the heme on a subnanosecond time scale. Sperm whale Mb and derivatives have two tryptophans and their decays can be interpreted mainly as two exponentials, one of ca. 20 ps and the other of 130 ps, whereas tuna Mb has one tryptophan and its emission is nonexponential but dominated by one component of 31 ps. These results along with F6rster energy transfer calculations allow us to assign the ca. 30-ps emission to Trp-14 and the 130-ps emission to Mb. The streak camera was modified to determine the decay of the fluorescence anisotropy. In metMb (tuna) the fluorescence anisotropy decays in 100 ps, which is postulated to result from rapid motion of the Trp-14. Because energy transfer was used to gate the anisotropy, the fast motion of Trp-14 is proposed to correspond to only 10% of the equilibrium distribution of molecules.Tryptophans are useful optical probes of protein structure because of their relatively long wavelength absorption compared with polypeptides and other common amino acid residues. A first step in realizing the full potential of such probes is the development of techniques to distinguish the different tryptophans in a protein by means of their optical responses. We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1-6), 02 (1,(4)(5)(6), and NO (6, 7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.Pulsed laser techniques are needed to study tryptophan fluorescence in heme proteins as a result of very efficient energy transfer to the heme. The low fluorescence quantum yields for tryptophan in heme proteins (8) suggest that the corresponding lifetimes are in the picosecond range. Thus, to obtain direct information on the lifetimes and motional characteristics of such tryptophans it was necessary to devise experiments by which the fluorescence and its polarization could be measured with picosecond accuracy.Sperm whale (SW) Mb is known to contain the two tryptophan residues, Trp-7 and Trp-14 (9). Thus, it is expected that the emission will be dominated by two decays. The heme is considerably further from Trp-7 than from Trp-14 (20 A compared with 15 A) so that the effects of energy transfer on these excited states are expected to be different. Tuna Mb has an x-ray structure very similar to SW Mb (10) bu...
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