Magneto-Optic Spectroscopy of a Protein Tetramer Binding Two Exciton-Coupled Chlorophylls Joseph L. Hughes,* ,§ Reza Razeghifard, ‡ Mark Logue, ‡ Aaron Oakley, § Tom Wydrzynski, ‡ and Elmars Krausz § Contribution from the Research School of Chemistry, and Photobioenergetics, Research School of Biological Sciences, The Australian National UniVersity, Canberra, ACT 0200, Australia Received September 25, 2005; E-mail: hughes@rsc.anu.edu.au Abstract: In vitro chlorophyll (Chl) aggregates have often served as models for in vivo forms of long- wavelength Chl. However, the interaction of protein-bound Chl molecules is typically different than that occurring in solvent-based self-aggregates. We have chosen a water-soluble Chl-binding protein (WSCP) from cauliflower in order to help characterize the spectroscopic properties of Chl in a single well-defined native environment and also to study the pigment-pigment (exciton) interactions present in assemblies of this protein. WSCP forms tetrameric units upon binding two Chl molecules. We present the absorption, circular dichroism (CD), magnetic circular dichroism (MCD), and emission spectra at 1.7 K of recombinant WSCP tetramers containing either Chl a or Chl d. The spectroscopic characteristics provide evidence for significant exciton interaction between equivalent Chl molecules. Our simple exciton analysis allows an estimate of the molecular geometry of the dimer, which is predicted to have an “open sandwich”-type structure. We find that the ratio of the magnetic circular dichroism to absorption, ΔA/A, is substantially increased (∼60%) for Chl a in this system compared to its value in solution. This increase is in marked contrast to substantial reductions (>50%) of ΔA/A seen in solvent-based Chl aggregates and in photosynthetic reaction centers. Current theoretical models are unable to account for such large variations in the MCD to absorption ratio for Chl. We propose that spectroscopic studies of WSCP mutants will enable a fundamental understanding of Chl-Chl and Chl-protein interactions. Introduction Photosystems are (bacterio)chlorophyll-containing pigment- protein complexes responsible for efficient light-induced electron transfer in photosynthesis. Properties of (bacterio)chlorophyll (B)Chl molecules important for their effective function in photosystems, such as their excitation energies or redox potentials, can be strongly influenced by their interactions with the surrounding protein environment. 1 Chl molecules bind to proteins via a number of noncovalent interactions, and the accumulated specificity of such interactions dictate the precise relative orientations and separation of Chl molecules within a photosynthetic assembly. In addition to the Chl-protein van der Waals interactions, protein residues typically provide a fifth ligand to the central metal (Mg) of Chl, 2,3 as well as giving rise to hydrogen bonding to the peripheral groups of the chlorin ring. 2 Binding is highly specific as structurally similar Chl molecules, such as Chl a and Chl b, cannot easily be interchanged. By modifying the protein site, the selective binding can be altered. 4 The photooxidizable assembly found in the reaction center of natural photosystems is often considered to be an exciton- coupled dimer and has been the subject of extensive spectro- scopic studies 5,6 to better understand its unique functionality. However, it can be a challenging task to study the optical spectroscopy of two particular Chl molecules in a photosystem that may contain up to several hundred Chl as well as other pigments. To exacerbate this problem, in general, each specific Chl-binding protein site will bind Chl molecules with different excitation energies and will exhibit an intrinsic inhomogeneous spectral broadening. This will lead to a spectrum consisting of overlapping bands that is difficult to characterize. Consequently, the study of a well-defined, isolated Chl dimer bound in a protein environment becomes appealing. In this study, we have chosen a water-soluble Chl-binding protein (WSCP) from cauliflower, which has a very high affinity for Chl a. 7 This protein is capable of extracting Chl from the proteins in thylakoid membranes in aqueous suspensions, forming Chl-WSCP. The WSCP class of proteins is different from Chl-containing membrane-bound proteins. They are water- soluble, contain high -structure content, and exhibit high § Research School of Chemistry. ‡ Photobioenergetics, Research School of Biological Sciences. (1) Renger, T.; Marcus, R. A. J. Chem. Phys. 2002, 116, 9997-10019. (2) Lutz, M.; Ma ¨ntele, W. Vibrational Spectroscopy of Chlorophylls. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1991; pp 855- 902. (3) Balaban, T. S.; Fromme, P.; Holzwarth, A. R.; Krauss, N.; Prokhorenko, V. I. Biochim. Biophys. Acta 2002, 1556, 197-207. (4) Bassi, R.; Croce, R.; Cugini, D.; Sandona `, D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10056-10061. (5) Dekker, J. P.; van Grondelle, R. Photosynth. Res. 2000, 63, 195-208. (6) Raszewski, G.; Saenger, W.; Renger, T. Biophys. J. 2005, 88, 986-998. (7) Satoh, H.; Nakayama, K.; Okada, M. J. Biol. Chem. 1998, 273, 30568- 30575. Published on Web 02/28/2006 10.1021/ja056576b CCC: $33.50 © 2006 American Chemical Society J. AM. CHEM. SOC. 2006, 128, 3649-3658 9 3649