7945BLM-Fe(II1) complexes and their 1:l cyanide adducts at 25 OC. The small proton paramagnetic shifts of the BLM-Fe(II1) complex (1 1.6 ppm) and the BLM-Fe(III)-CN3NH2 complex (25.1 ppm) indicate low-spin ferric (S = state. Indeed, the ESR features of these Fe(II1) complexes at 77 K are characteristic of a rhombic low-spin type: g, = 1.893, gy = 2.185, and g, = 2.431 for the BLM-Fe(II1) complex and g, = 1.847, gy = 2.179, gr = 2.540 for its methylamine a d d u~t .~ On the other hand, the addition of cyanide ion to the BLM-Fe(II1) and BLM-Fe(II1)-CH3NH2 complexes produced a drastic change in the original 'H N M R spectra, and new proton peaks appeared in the lower and higher field regions. The magnitude of the chemical shifts (over f50 ppm) strongly suggests a high-spin ferric type for these cyano complexes.* CO, NO, and C2H5NC bind to the BLM-Fe(I1) complex as a sixth ligand to form low-spin ferrous adduct complexes,2 and several nitrogenous bases also coordinate to the BLM-Fe(II1) complex to give low-spin ferric adducts.5 If C N ion similarly binds to the vacant sixth coordination site of the BLM-iron complex, which has a rigid square-pyramidal arrangement with the 5-5-5-6 ring member,5 the cyanide adducts would be expected to have a low-spin iron state.We have found that the BLM-Fe(II)-02 system efficiently produces oxygen radicals such as 02-* and -OH.9 Indeed, similar ESR spin-trapping experiments using N-tert-butyl a-phenyl nitrone have shown that the addition of CO (or C2HSNC) strongly interferes with O2 activation by the BLM-Fe(I1) complex, but stoichiometric C N addition slightly increases the production of oxygen radicals in comparison with the original BLM-Fe(I1) system.BLM-iron complexes and hemoproteins apparently display similarities in the binding of oxygen antagonists (CO, NO, and C2H5NC) and in external nitrogenous bases, but the interaction of C N ion is remarkably different. In general, cyanide interferes with reaction of heme oxygenases, and C N adducts of ferric hemoproteins are of low-spin type. The present unusual behavior of C N ion toward the BLM-iron complexes appears to be responsible for the cyanide enhancement of the BLM activity against DNA. A detailed investigation of the C N interaction and the complete assignment of the proton signals are now under way.Gratitude is due to Professor Hamao Umezawa for encouragement, Dr. Tomohisa Takita for pertinent advice, and M. Ohara for comments on the manuscript. This study was supported in part by a grant from the Ministry of Education, Science, and Culture, Japan. Acknowledgment.(8) When CN ion was added to BLM-Fe(II1) and its CH3NH2 adduct complexes, the typical low-spin ESR signals disappeared and a new broad ESR absorption near g = 4 appeared. However, quantitative consideration of this signal is difficult at present because of its complexity.(9) (a) Sugiura, Y.; Kikuchi, T. Sir:We proposed the external point-charge model I (Figure 1)1-3 to account for the variance in the absorption maxima of various (1) Nakanishi, K.; Balogh-Nair, V.; Gaw...

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Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position or intensity between rhodopsin and the photoproducts formed at low temperature (70 K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in the identification of the vibrational modes, we performed experiments on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13C at carbon 15 or 15-deuterioretinal. Our infrared measurements provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is distorted all-trans. The positions of the C = N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis-15-deuterioretinal or 11-cis-retinal containing 13C at carbon 15 are indicative that the Schiff-base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C = N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C = N trans conformation in all three species. Finally, we present evidence that, even in these early stages of the rhodopsin photosequence, changes are occurring in the opsin and perhaps the associated lipids.

We studied the nature of the protein binding site of rhodopsin, using two-photon spectroscopy to assign the location of the low-lying "covalent" A *.-like 1rir* state in a model rhodopsin containing a locked-li-cis chromophore. The two-photon thermal lens maximum is observed at 22,800 cm-, "2000 cmt above the one-photon absorption maximum, is also proposed. The latter model is interesting because it also accommodates the observed deuterium isotope effect in the form of a proton translocation between the two residues. The translocation is assumed to be a ground state process, initiated subsequent to the photoisomerization of the chromophore and energetically driven via destabilization of the counterion environment as a result of isomerization-induced charge separation.The nature of the protein binding site of rhodopsin is a subject of intense study (1-18) and continued debate (for recent reviews see refs. [1][2][3][4][5]. Despite the efforts of many researchers, well-ordered three-dimensional crystals of rhodopsin have not been prepared, precluding structural analysis using high resolution x-ray diffraction. Thus, the majority of experimental studies have utilized electronic absorption (6-10), resonance Raman (11,12), nuclear magnetic resonance (13), Fourier-transform IR (14-16), or picosecond spectroscopy (17, 18). The above-cited experimental, as well as theoretical (19-24), investigations are not in general agreement concerning the state of protonation of the chromophore (see, for example, refs. 2, 3, 5-8, 11-16, and 18-24) or the number and location of the counterions inside the binding site (2, 6-8, 18-21, 23). With the goal of resolving some of these issues, we report here the two-photon spectrum of locked-ilcis-rhodopsin.The unique diagnostic capabilities of two-photon spectroscopy for studying the binding site of rhodopsin derive in part from the unusual electronic properties of polyenes. [The polyene chromophore of rhodopsin is 11-cis-retinal bound to the opsin protein via a covalent linkage with the E-amino nitrogen of lysine (1)(2)(3)(4)(5) (27). The latter method, which is described in this paper, proved to be more reliable and is based on the previous synthetic work of Akita et al. (8). The incorporation of the locked-11-cis chromophore (shown in Fig. 1) into opsin produces a nonbleachable rhodopsin analog that can withstand the high light fluxes used in two-photon spectroscopy (9, 27). There is compelling evidence to suggest that the "locked" chromophore occupies the same binding site as the native 11-cis chromophore, and the spectroscopic (one-photon absorption and CD) similarities suggest that the counterion environment is unperturbed (8).The following sections describe the generation of the twophoton thermal lens spectrum and the theoretical analysis of the spectroscopic data. Our principal goal is to assign the state of protonation of the chromophore and determine the nature of the local counterion environment. 4117The publication costs of this article were defrayed in part by page c...

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