J. Phys. Chem. 1990, 94, 7927-7935
7927
these cations to the metal binding sites, causing the excess heat capacity curves to be representative of sequential melting involving states N1, N2, and D. The order of stabilization of the N1 state by binding to cations is Li+ > Na+ > K+ >> tetraethylammonium (see Table 1 for transition temperatures at 12 mM), but the same order of binding affinities exists for the N2 state because the transition from state N2 to D depends on which cation is present, e.g., 49.2 “ C for tetraethylammonium and 59.5 O C for Li+, both at 12 mM. Breaking calmodulin in the middle or clipping off the fourth metal binding region produces proteins that melt, according to Tsalkova and P r i v a l ~ v via , ~ ~an apparent two-state process, which is consistent with the entire calmodulin molecule being involved in the final transition. Addition of ethanol to calmodulin solutions (1 2 mM K+) moves the entire transition region to lower temperatures, but the change is about 6 times what would be expected from predictions based on studies by Sturtevant and V e l i ~ e l e bon i ~ ~lysozyme (1 29 amino acid residues and a single two-state transition). All calmodulin’s a-helices have a net hydr~phobicity,~~ in spite of a regular in-
terspersion of polar and hydrophobic amino acid residues, which is unusual in a small protein. Thus, the large shift in ethanol solutions is probably due to calmodulin’s hydrophobicity; Le., each a-helix will be less stable in the ethanol-water environment because the denatured state is favored via solvation of the hydrophobic residues by ethanol. The very large slope seen in the excess heat capacity curves for the native protein (also found by Tsalkova and P r i v a l ~ v ~ ~ * ~ ~ ) appears to be characteristic of calmodulin’s dumbbell shape, which would be expected to have many low-frequency vibrations that would contribute substantial changes to the excess heat capacity curve. Calmodulin has a large number of hydrophobic residues, but the excess heat capacity actually decreases with increasing t e m p e r a t ~ r efor ~ ~small apolar solutes which might be models for these residues. Thus, the unusual slope seen for calmodulin is unlikely related to its hydrophobicity. Registry No. Et, 64-17-5; N(Et),+, 66-40-0; Li, 7439-93-2; Na, 7440-23-5;K, 7440-09-7;Ca, 7440-70-2;Mg, 7439-95-4; Mn, 7439-96-5; Sr, 7440-24-6; Cd, 7440-43-9;Co, 7440-48-4.
(48) Cox, J. A,; Comte, M.; MalnGe, A. In Metal Ions in Biological Sysfems;Segiel, H., Ed.; Marcel Dckker: New York, 1984; Vol. 17.
(49) Gill, S.J.; Dec, S.F.; Olofsson, G.; Wadso, 1. J . Phys. Chem. 1985, 89, 3758.
Characterization of Ground and Electronically Excited States of o-Hydroxybenzaldehyde and Its Non-Hydrogen-Bonded Photorotamer in 12 K Rare Gas Matrices Meredith A. Morgan,+ Edward Orton,* and George C. Pimentelt Laboratory of Chemical Biodynamics, University of California. Berkeley, Berkeley, California 94720 (Received: January 30, 1990: In Final Form: May 3, 1990)
Intramolecularly hydrogen bonded o-hydroxybenzaldehyde (OHBA-C) isblated in 12 K rare gas matrices photolyzes to a non-hydrogen-bonded rotamer (OHBA-F). IR spectra of OHBA-C, OHBA-F, and several model and isotopically substituted compounds are consistent with identification of the OHBA-F conformer as that formed by 180-deg rotation of both the hydroxy and aldehyde groups. For the two rotamers, electronic absorption, excitation, and emission spectra are presented together with time-resolved emission measurements and estimates of a ground-state reaction enthalpy. From these data, it is proposed that the S, state of OHBA-C is an n,r* hydrogen atom transfer state, and Sz is a r,r* proton-transfer state with a large (- 18 kcal) barrier to reaction. Rotamerization is reversed by SI or S2excitation of OHBA-F. The conversion of OHBA-C to OHBA-F is - 5 times as efficient as the reverse process upon excitation at the respective SI 0-0 energies. An increase in photolysis quantum yield of OHBA-C is measured at energies well above the 0-0 energy and may correspond to reaction over the proposed -8 kcal SI barrier.
Introduction The spectroscopy of o-hydroxybenzaldehyde (OHBA-C in Figure 1) has been actively investigated for many years. Structurally, it is the simplest of the intramolecularly hydrogen bonded salicylic acid derivatives which are thought to undergo proton or hydrogen-atom transfer in the S, state. It remains an area of debate which of these transfer processes takes place in the SI state, and whether SIis n,r* or r,r*.’s2OHBA-C forms a non intramolecularly hydrogen bonded rotamer upon UV photolysis in cryogenic mat rice^.^ The precise structure of the non-hydrogen-bonded conformer has not been resolved.’ Previous studies have been hampered by reliance on either emission or infrared absorption spectroscopy alone, as well as by complications from intermolecularly hydrogen bonding solvents or impurities. Proton-transfer systems are of interest for four-level lasers, and photochemically reversible isomerizations are of interest as prototypes for molecular information or energy storage systems. ‘Author to whom correspondence should be addressed. ‘Current address: Rohm and Haas Co., Architectural Coatings Research, Spring House, PA 19477. *Deceased June 18, 1989.
0022-3654/90/2094-7927$02.50/0
In this study of OHBA in cryogenic rare gas matrices, we identify the conformation of the non intramolecularly hydrogen bonded rotamer from high-resolution infrared spectra. We also present high-resolution steady-state and time-resolved emission spectra of OHBA-C and its photorotamer in argon and xenon matrices. From these spectra we derive information on the excited states of both conformers: 0-0 energies; vibrational modes active in the chromophores; and the electronic configurations of SI and S2. Combining these data with the vibrational structure in the rotamer’s excitation spectrum, and with ground-state enthalpiese6 and barriers to reaction4*’measured and estimated by others, we ( 1 ) Nagaoka, S.;Hirota, N.; Sumitani, M.; Yoshihara, K. J. Am. Chem. SOC.1983, 105, 4220. (2) Nagaoka, S.; Nagashima, U.; Ohta, N.; Fujita, M.; Takemura, T. J . Phys. Chem. 1988, 92, 166. (3) Gebicki, J.; Krantz, A. J . Chem. Soc., Perkin Trans. 2 1984, 1617. (4) Tabei. M.: Tezuka. T.: Hirota. M. Tetrahedron 1971. 27. 301. ( 5 ) Rajogopal, E.; Sivakumar, K. V.; Subrahmanyam, S.V. J . Chem. SOC., Faraday Trans. I 1987, 77, 2149. (6) Schaefer, T.; Sebastian, R.; Laatikainen, R.; Salman, S. Can. J. Chem. 1984, 62, 326. (7) Anet, F. A. L.; Ahmad, M. J . Am. Chem. SOC.1964, 86, 119.
0 1990 American Chemical Society
1928 The Journal of Physical Chemistry, Vol. 94, No. 20, 1990
I
OHBA-C H
U
"\
0
0 I1
11
\
O
H
r
111
Figure 1. Molecular structures of OHBA-C and plausible conformers (1-111)
for its photorotamer, OHBA-F.
are able to estimate the depth of OHBA-C's excited-state potential wells. We have an ongoing interest in this laboratory in excited-state reaction pathways. However, the potential surfaces of most photochemically reactive molecules have not been mapped in detail. In an experiment analogous to measurements of ground-state reaction barriers from the wavelength dependence of infrared-induced reactions in cryogenic rare gas matrices,8 we have measured the wavelength dependence of the reaction quantum yield for the laser photolysis of OHBA-C to its rotamer. An increase in the reaction quantum yield at higher photolysis energies may be indicative of reaction over an SI barrier. Rare gas matrices are the preferred media for such studies, since the phonon modes of crystalline rare gases tend to couple poorly to vibrational modes of guest molecules. Thus, fluorescenceg and reaction* are more competitive with vibrational relaxation in these matrices. The photolysis of OHBA is quite facile in both the forward and reverse directions, even with excitation at the SI0-0 energies. We have measured the ratio of the quantum yields for the forward and reverse reactions in both argon matrix and the triplet-directing heavy atom matrix of xenon. These data, in conjunction with excited-state assignments and lifetimes, are indicative of the reaction pathway(s) accessible below the s, barrier. Thus, with information from vibrational and electronic absorption, steady-state excitation and emission, and time-resolved emission spectroscopies, tuned laser photolysis, and variation of the spin-orbit coupling ability of the matrix, both the contours of the electronic potential surfaces and the reaction dynamics on those surfaces are better understood. Experimental Section Instrumentation. Instruments used for acquisition of cryogenic matrix IR absorption spectra, steady-state emission spectra, and fluorescence lifetimes have been described previously.I0 The excitation filter used for the fluorescence lifetime was a Corning 7-37 colored glass filter (320 < X < 400 nm), while emission light was selected with a Corning 4-65 colored glass filter (460 < X < 600 nm). The instrument response function was generated by replacing the excitation filter with another Corning 4-65 filter and attenuating the signal with neutral-density filters. The phosphorescence lifetime of OHBA-C's photoproduct was measured with a Spex 212T steady-state fluorometer in conjunction with a fast electronic shutter (Uniblitz Model 100-2B, A. W. Vincent Assoc., Inc.). The emission PMT signal decay was recorded on a digital oscilloscope (Explorer IIIA, Nicolet Instrument Corp.) triggered by the shutter. The decay curve ( 5 ps/channel) was subsequently plotted on an x-y plotter and the process repeated three times to reduce noise. Discounting the first ~
~
~~
Morgan et al. millisecond of decay, encompassing the time of shutter closure, the decay curve was analyzed by a nonlinear least-squares fitting program, assuming a single lifetime. The lifetime was determined with excitation at 290 nm and emission at 404 nm (4 nm bandpass). UV absorption spectra were collected with a Shimadzu 2100 UV/vis spectrometer, at 0.1-nm resolution. The UV spectrum of OHBA-C's photolysis product was collected following irradiation of the OHBA-C matrix for 10 min with a 1000-W high-pressure Hg-Xe lamp through a 6-in water filter and a 7-60 glass filter (300-380 nm, Corning). IR samples were photolyzed with the frequency-modified output of a DCR Nd:YAG-pumped (Quanta Ray) dye laser (Model PDL-I, Spectra Physics), fitted with filled-in doughnut optics. The dye laser was operated in its end-pumped amplifier configuration to maintain a circular output. The dye laser output was selected with a grating, precise to f0.001 nm. The wavelength of the visible dye laser output was calibrated to f O . l nm with a I-m single monochromator (Model MP-1018, McKee-Pederson Instruments), which was in turn calibrated with an Hg pen lamp. A wavelength extension system (Model WEX-1, Quanta Ray) was used to either double the dye laser output to access wavelengths in the region 280-330 nm (Rhodamine 590, 610, and 640, Kiton Red, and DCM dyes, Exciton), or to add the 1.06-wm fundamental output of the Nd:YAG to access the 366-407-nm region. Photolysis powers were usually maintained at
