Determination of the Chromophore Structures in the Photoinduced Reaction Cycle of Phytochrome Maria Andrea Mroginski,* ,† Daniel H. Murgida, † David von Stetten, † Christa Kneip, ‡ Franz Mark, ‡ and Peter Hildebrandt* ,† Technische UniVersita ¨t Berlin, Institut fu ¨r Chemie, Sekr. PC 14, Strasse des 17. Juni 135, D-10623 Berlin, Germany, and Max-Planck-Institut fu ¨r Bioanorganische Chemie, Stiftstrasse 34-36, D-45470 Mu ¨lheim, Germany Received October 4, 2004; E-mail: hildebrandt@chem.tu-berlin.de Phytochromes constitute a family of sensory photoreceptors that are ubiquitous in plants and have been recently also discovered in bacteria. 1 The chromophoric site is constituted by a linear methine- bridged tetrapyrrole (Figure 1) that is covalently attached to the apoprotein. Upon light absorption, phytochrome runs through a reaction sequence from the inactive form P r to the active form P fr triggering the signal transduction pathway. It is commonly accepted that the photoprocess involves a Z f E isomerization of the methine bridge between the rings C and D. 2 However, the three-dimensional structure of phytochrome is not yet available, and even for the stable states sound information about details of the chromophore structure has not been obtained thus far. In this respect, resonance Raman (RR) spectroscopy is an indispensable technique since it exclusively probes the vibrational bands of the tetrapyrrole. However, there is no consensus on the interpretation of the RR spectra that have been obtained for the parent states and the photocycle intermediates. 3,4 In this contribution we present a combined experimental and theoretical approach to determine the phytochromobilin (PΦB) structure from the RR spectra of phytochrome phyA (oat) by comparison with Raman spectra calculated for different tetrapyrrole geometries. Vibrational spectra were obtained by density functional theory (DFT) using the B3LYP functional 5 and the 6-31G* basis set. The force field was scaled by a set of global scaling factors determined for a series of model compounds including hydrogen- bonded systems. 6 This approach affords an accuracy of (11 cm -1 for the calculated frequencies. Calculated Raman intensities that serve as additional assignment criteria agree qualitatively with the experimental data 6 and, moreover, provide a good description for the experimental spectra of tetrapyrroles measured with preresonant excitation. 4b,7 For open-chain tetrapyrroles, the number of isomers that differ with respect to the methine bridge configuration (Z/E) and conformation [syn(s)/anti(a)] is 64 (Figure 1). In 32 geometries, the C-D methine bridge is in the Z configuration as it is most likely to be in the P r state. 2 Furthermore, the P r chromophore probably exhibits an extended structure. 8 Thus, we have sorted out isomers with largely helical structures and isomers exhibiting highly distorted geometries due to steric interactions. The remaining 15 isomers that include also those previously proposed for the chromophore structure in P r , 3,4,7 constitute the set of geometries for the DFT calculations. In each of these methine bridge isomers, the side chains of the pyrrole rings can adopt different conforma- tions. As analyzed for a few cases, different propionic side-chain conformations are reflected in the Raman spectrum only below 1000 cm -1 , whereas variations of the methine bridge isomerization affect the spectrum also and quite markedly between 1000 and 1700 cm -1 (vide infra). Also esterification of the propionic acid side chains caused only small spectral changes exclusively in the low frequency region. For all further calculations the methyl ester forms were used to avoid intramolecular hydrogen-bond interactions in the tetrapy- rrole, which most likely do not exist in phytochrome. Significant interactions with the protein are expected for the N-H groups that are likely to form hydrogen bonds with adjacent amino acid residues. Since RR spectroscopy has revealed that all nitrogens are protonated in each state of phytochrome detected thus far, 4 a counterion has been included in the calculations. In phytochrome, the counterion has not yet been identified, but it is likely to be a carboxylate group. However, calculations with acetate and Cl - ion yield very similar Raman spectra such that we have used a Cl - ion as the simpler variant in all calculations. Finally, the thioether function (ring A), which constitutes the covalent linkage to the protein, is replaced by an ethyl group in the calculations. Using these approximations, we have optimized the geometries of more than 20 methine bridge isomers. For calculating the Raman spectra, standard scaling factors were employed 6a except for the N-H internal coordinates for which the scaling factors of the hydrogen- bonded hexamethyl pyrromethene monomer were adopted. 6b Experimental RR spectra of the various states of phytochrome phyA (oat), which exhibit absorption maxima between 660 and 720 nm, were obtained with 1064-nm excitation at low temperature as described previously. 4 Under these preresonance conditions, Raman signals of the apoprotein only contribute to broad and weak humps in the spectrum. In the region above 1000 cm -1 , the Raman spectrum of the ZZZasa isomer agrees very well with the experimental RR spectrum of P r as shown for the range between 1490 and 1700 cm -1 in Figure 2. Thus, all of the observed RR bands can readily be assigned to the calculated bands. Relative intensities are well reproduced, and the frequency deviations are within the error found for model compounds. 6 This is also true for the chromophore deuterated at the pyrrole nitrogens (Figure 2). For all other methine bridge isomers, the agreement with the experimental spectra is much worse as illustrated for the ZEZaas geometry which has been previously proposed to be the chromophore conformation in P r (see Supporting Information). 3b Hence, we conclude that in P r the chromophore is in the ZZZasa configuration, thereby confirming previous sugges- tions. 4,7 The results further reveal that the frequency range between † Technische Universita ¨t Berlin. ‡ Max-Planck-Institut fu ¨r Bioanorganische Chemie. Figure 1. Phytochromobilin (PΦB) in the ZZZasa geometry. Published on Web 12/02/2004 16734 9 J. AM. CHEM. SOC. 2004, 126, 16734-16735 10.1021/ja043959l CCC: $27.50 © 2004 American Chemical Society