Solvation dynamics around a solute dissolved in water have been measured for the first time on the femtosecond time scale. The measurements were made by the fluorescence time-dependent Stokes shift technique, which employs polar fluorescent probes and ultrafast fluorescence spectroscopy to characterize the solvation dynamics of electronically excited molecules.The probe used in this study is the 7-(dimethylamino)coumarin-4-acetate ion. The observed microscopic solvation dynamics are well modeled by a biexponential decay with lifetimes ( ,, t2) and amplitudes (A¡, A2) as follows: t¡ = 0.16 ps (A¡ = 0.33) and r2 = 1.2 ps (A2 = 0.67). The experimental results have been compared to predictions from the dielectric continuum theory, the linearized MSA theory, and recently published molecular dynamics simulations of water solvation.

in part to a change in configuration to a tetrahedral geometry cannot be determined from these results. Such evidence may be gleaned from future high-temperature spectrophotometric, Raman, NMR, or neutron-scattering experiments.Another consideration for establishing the configuration of the acetate complexes concerns whether this ligand is mono-or bidentate in character. Numerous X-ray crystallographic and spectroscopic data indicate that in the solid state acetate may serve as a bridging ligand,29•30 a bidentate ligand,31-34 a monodentate ligand with partial secondary bonding,35 and a mondentate ligand.29,36•37 However no corresponding results were found for ferrous complexes, and it cannot be assumed that the same bonding will necessarily be preserved in solution. Certainly at 25 °C the formation constants for ferrous complexes with known bidentate ligands such as oxalate and malonate (log X, = 3.02 and 2.24, respectively)38 are somewhat larger than for acetate (1.439), but this cannot be construed as definitive evidence for either mono-

The transient solvation of a polar fluorescent probe has been studied by the time resolved Stokes shift technique with roughly five times shorter time resolution than previously reported. New shorter time components in the solvation relaxation function C(t) have been discovered for methanol, propionitrile, and propylene carbonate; the C(t) function for acetonitrile is singly exponential within the limitations of the instrument. The observed C(t) has been compared to theoretical calculations using the dielectric continuum (DC) model for each solvent, with non-Debye expressions for the solvent dielectric response. For methanol the DC model predictions agree closely with experiment. For the polar aprotic solvents propylene carbonate and propionitrile, the shape of the experimental decay is different from the DC predictions, but the average decay times 〈τs〉 are closer to the DC predictions than previously reported. The comparison of theory and experiment is clearly limited by the inconsistencies and limited frequency range of the dielectric relaxation data found in the literature. The dynamic solvation measurements have also been compared to predictions of the mean spherical approximation as applied to solvation dynamics, which appear to give slower solvation rates than are observed experimentally.

It has been found that the electron-transfer time ret of the intramolecular electron-transfer reaction of electronically excited bianthryl is not equal to the longitudinal relaxation time of the solvent in various polar aprotic solvents. This demonstrates that the recently observed agreement of ret and r¡, in specific cases, is not general. In addition, microscopic solvation times ts have been measured for the solvents, and it has been observed that t, is very similar to for a broad range of polar aprotics.

Surface science model studies of Cs solvation by protic and aprotic solvents J. Vac. Sci. Technol. A 4, 1307 (1986); 10.1116/1.573598Snowballing effect for electron solvation in dilute solutions of polar molecules in nonpolar solvents Subpicosecond fluorescence spectroscopy of the polar dye molecules coumarin 311 and coumarin 102 has been used to measure the microscopic solvation dynamics of several polar aprotic solvents. The measured solvation times are significantly longer than the longitudinal relaxation times 1"1 ofthe solvents. 1"1 is the predicted time for solvation according to dielectric continuum theory. The experiments were made with a newly constructed subpicosecond ultraviolet emission apparatus that takes advantage of recent advances in ultrafast laser technology. The newly developed, time saving procedure [Nagarajan et al., J. Chem. Phys. 86, 3183 (1987)] for measuring the microscopic solvation response functions was used in this research.2372

We have built a new apparatus to time resolve ultrafast fluorescence following ultraviolet excitation. A synchronously pumped dye laser produces optical pulses of 1-ps or 70-fs full-width half-maximum, depending upon dyes and optical configuration. These pulses are amplified at a 8.2-kHz repetition rate using a copper vapor laser-pumped multipass amplifier. The resulting amplified laser pulses are frequency doubled to obtain ultrafast pulses in the ultraviolet. This ultraviolet light is used to electronically excite a sample; the resulting fluorescence is time resolved using fluorescence upconversion as the optical gating technique. A minimum 300-fs full-width half-maximum instrument response function is obtained. After a brief introduction, we discuss the principles involved in this method of time resolving fluorescence. We review special considerations for femtosecond laser experimentation, and discuss the construction of our apparatus. Finally, as an example, we show how this system can be used to study fundamental physical processes in solution.

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectra...

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.