Influence of Structure on Binding of Chlorophylls to Peptide Ligands Min Chen, ‡ Laura L. Eggink, § J. Kenneth Hoober,* ,§ and Anthony W. D. Larkum ‡ School of Biological Sciences, UniVersity of Sydney, NSW 2006, Australia, and School of Life Sciences and The Center for Early EVents in Photosynthesis, Arizona State UniVersity, Tempe, Arizona 85287-4501 Received October 28, 2004; E-mail: khoober@asu.edu Chlorophyll (Chl) is the essential pigment for oxygenic photo- synthesis in cyanobacteria, algae, and plants. Chl occurs as four different species among these organisms, and each binds in a highly specific and ordered manner to proteins. Until recently, Chl a (see Figure 1 for structures) was thought to be the pigment in reaction centers in all of these organisms. 1 The accessory Chls, Chl b and Chl c, reside in specific sites in light-harvesting complexes (LHCs), 2 as exemplified by LHCII associated with photosystem II in plants. 3 In 1996, Miyashita et al. 4 discovered a cyanobacterium (Acaryochloris marina) in which over 95% of the Chl is Chl d. Chl d not only is the major light-harvesting pigment in A. marina but also occurs in reaction centers. 5 In this study, we addressed the role of modifications at the periphery of Chl molecules on the chemistry of their interactions with protein-bound ligands. Introduction of the electronegative 3-formyl group in Chl d extends the electronic distribution along the Q y axis, shifts the absorbance maximum to longer wavelengths, and increases the absorption coefficient as compared with those of Chl a. The 7-formyl group of Chl b withdraws electrons toward the periphery of the molecule in the Q x direction, which reduces the dipole strength. 6 Consequently, the long-wavelength maximum of the spectrum is blue-shifted and the absorption coefficient is reduced relative to those of Chl a. This effect is intensified in Chl c, in which the C17-C18 double-bond of protochlorophyllide is retained and the ring conjugation system is extended to the unesterified, electronegative side-chain carboxyl group by introduc- tion of the C17 1 -C17 2 trans double bond. 7 The Mg atom in Chl is pentacoordinate, with four ligands provided by the tetrapyrrole nitrogens. The fifth, axial ligand is provided by an amino acid side chain in a protein or water. It was proposed that the electronegative character of the 7-formyl group of Chl b and the trans-acrylate group of Chl c increases the Lewis acid strength of the Mg atom relative to Chl a, thereby requiring harder Lewis bases in proteins to displace a tightly bound water ligand. 8 These structural variations led to the question of whether the coordination chemistry of Chl d would be similar to that of Chl a or Chl b. Eggink and Hoober 9 designed a synthetic peptide containing the sequence NH 2 -GLLAWRSHIVELAAGG-CONH 2 , which was adapted from Chl-binding sites in the LHCII apoprotein (LHCP). Each of the Glu(E)-Arg(R) ion-pair and His(H) ligands binds a molecule of Chl a, as assayed by Fo ¨ rster resonance energy transfer from the adjacent Trp(W) residue to bound Chl. This assay was used to compare the ability of these ligands to interact, in addition, with Chls b, c, and d. -n-Dodecyl maltoside (1 mM; critical micelle concentration, 0.18 mM) generated micelles that simulated a membrane environment. 10 The number of detergent molecules per micelle has been reported as 78-92 (Anatrace, Inc.) or 110-140, 11 which provided a micelle-equivalent concentration in the range of 8-10 µM. A 1 mM solution of the peptide was added with stirring to the reaction mixture containing 100 nM Chl, 12 buffered with 50 mM Na borate, pH 9.0, and the sample was allowed to equilibrate at 37 °C for 15 min after each addition prior to spectral analysis. The Chls have nearly equal absorbance (excitation) maxima between 330 and 350 nm (Figure 2) that overlap the emission spectrum of Trp, a requirement for energy transfer. 9 When peptide ‡ University of Sydney. § Arizona State University. Figure 1. Structures of Chls a, b, c, and d. The esterified phytol on position 17 3 is shown in the structure of 3-monovinyl Chl a. Also shown are the Qy and Qx axes of the molecule. Chl b is identical to Chl a, except for oxidation of the 7-methyl group to a 7-formyl group. Chl c retains the C17-C18 double bond of the precursor protochlorophyllide and includes an additional double bond between C17 1 and C17 2 . The 17 3 -carboxyl group remains unesterified. Chl c1,R ) C2H5; Chl c2,R ) C2H3. Chl d is identical to Chl a except for oxidation of the 3-vinyl group to a formyl group. Ph ) phytol. Figure 2. Excitation spectra of the Chls with various concentrations of the peptide. (A,D) Peptide was added to Chls a and d, respectively, to concentrations of 0, 5, 10, and 20 µM. (B,C) Peptide was added to Chl b to concentrations of 0, 10, 30, and 60 µM and to Chl c to 0, 20, 40, and 60 µM. Numbers under the peak at 280 nm refer to peptide concentration. Published on Web 01/26/2005 2052 9 J. AM. CHEM. SOC. 2005, 127, 2052-2053 10.1021/ja043462b CCC: $30.25 © 2005 American Chemical Society