Hydrophobic interactions in metalloporphyrin-peptide complexes.

The study of synthetic analogues of heme proteins has been thorough and successful.1 Despite many accomplishments, however, there remains a fundamental difference between the heme proteins themselves and the small molecules that mimic them; the analogues are generally not made from peptides, and the interactions between the heme and the protein remain largely unexplored.2 The few exceptions are small covalently linked heme-oligopeptides digestion products derived from cytochrome c,3 short oligopeptides covalently linked to the periphery of porphyrins or coordinated to exchange-inert metalloporphyrins,4 the elegant helical bundle peptide-heme complexes of DeGrado, Dutton, and co-workers,2,5 and our recent work on amphiphilic peptide-metalloporphyrin complexes.6 Studies of this “mesomolecular” regime (i.e., from roughly 1000 to 10 000 amu) represent an emerging field that has only recently become synthetically and analytically accessible. In this paper, the direct ligation of polypeptides in synthetic heme-peptide complexes has been characterized. To this end, we have examined the influence of peptide length, sequence, and properties on their complexation to a water-soluble Fe(III) porphyrin. Recent success in the design of peptides2 has provided insight into the design of synthetic proteins. Synthetic heme proteins require additionally the integration of a ligating residue into the peptide sequence and the establishment of favorable nonbonding interactions between the metalloporphyrin and the peptide. In addition to metal ligation of amino acid residues, secondary nonbonding interactions may also be important because the heme is sandwiched tightly between two or more helices.7 Binding of series of peptides8 by the FeIII complex of coproporphyrinate-I (3,8,13,18-tetramethyl-2,7,12,18-tetrapropionateporphyrinate, copro) was determined by spectrophotometric titration.9 FeIII(copro) was chosen because it has a symmetrical distribution of four carboxylates, which increases its solubility and substantially decreases aggregation. Ligand binding studies indicate in all cases that only 2:1 peptide-metalloporphyrin complexes are formed with bisimidazole coordination. The data are shown in Table 1 and Figure 1. To probe the importance of hydrophobic interactions between the peptides and the porphyrin face, the effects of multiple alanine residues (a strong R-helix former)10 were examined. Histidine was utilized as the ligating residue in our peptides, analogous to the globins, peroxidases, and b cytochromes. Solubility of the peptides was provided by Glu or Ser residues and by not end-capping the peptides, thereby having charged amino and carboxy termini. Despite the possibility of helix-turn-helix motifs11 in our longest peptides,12 no clear examples of 1:1 binding were observed. In retrospect, this is perhaps not surprising because conformational demands strongly disfavor formation of 1:1 complexes. Relative binding constants increase by a factor of 1.6 × 104 as the peptide length increases. The cause of this dramatic increase in relative binding cannot be explained by the imidazole ligation to the metal center, which remains constant. Other factors that might contribute to the stability of the complexes include (1) electrostatic interactions between the porphyrin and the peptide, (2) hydrogen bonding between the porphyrin and the peptide, and (3) hydrophobic interactions between the porphyrin and the peptide. Electrostatic interactions are not likely to be favorable in these metalloporphyrin-peptide complexes, since the overall charge is negative for both the metalloporphyrin and the ligand for peptides 5-7. Hydrogen bonding will not explain the systematic trend observed in Table 1 and Figure 1. For the short sequences, hydrogen bonding is structurally untenable to the heme’s propionate side chains. For the longer sequences, if the peptides are helical when bound, again hydrogen bonding is precluded. In peptides 5-7, the asparagine or cysteine side chains may possibly hydrogen-bond to the propionate side chains of the metalloporphyrin, but this would not explain the overall trend. Both these peptides and the π-face of the metalloporphyrin are highly hydrophobic. We therefore suggest that hydrophobic interactions influence peptide binding to heme and are the primary factor responsible for the 1.6 × 104 increase in binding that we observe. As shown in Figure 1, as the number of hydrophobic residues increases, the binding constants increase substantially. The hydrophobic effect (i.e., gain in free energy on the transfer of nonpolar residues from an aqueous environment to a nonpolar environment) has provided a major unifying concept in under(1) (a) Collman, J. P. Inorg. Chem. 1997, 36, 5145. (b) Suslick, K. S. In The Porphyrin Handbook; Kadish, K., Smith, K., Guilard, R., Ed.; Academic Press: New York, 2000; Vol. 4, Chapter 28, pp 41-63. (2) (a) DeGrado, W. F.; Summa, C. M.; Pavone, V.; Nastri, F.; Lombardi, A. Annu. ReV. Biochem. 1999, 68, 779-819. (b) Tuchscherer, G.; Scheibler, L.; Dumy, P.; Mutter, M. Biopolymers 1998, 47, 63. (3) Jackson, A. H.; Kenner, G. W.; Smith, K. M.; Suckling, C. J. J. Chem. Soc., Perkin Trans. 1982, 1, 1441. (4) (a) Castro, B.; Gabriel, M.; et al. Tetrahedron 1981, 37, 1893, 1901, 1913. (b) Sasaki, T.; Kaiser, E. T. J. Am. Chem. Soc. 1989, 111, 380. (c) Karpishin, T. B.; Vannelli, T. A.; Glover, K. J. J. Am. Chem. Soc. 1997, 119, 9063. (d) Liu, D. H.; Williamson, D. A.; Kennedy, M. L.; Williams, T. D.; Morton, M. M.; Benson, D. R. J. Am. Chem. Soc. 1999, 121, 11798-11812. (e) Arnold, P. A.; Shelton, W. R.; Benson, D. R. J. Am. Chem. Soc. 1997, 119, 3181. (f) Sakamoto, S.; Sakurai, S.; Ueno, A.; Mihara, H. J. Chem. Soc., Chem. Commun. 1997, 1221. (5) (a) Robertson, D. E.; Farid, R. S.; Moser, C. C.; Urbauer, J. L.; Mulholland, S. E.; Pidikiti, R.; Lear, J. D.; Wand, A. J.; DeGrado, W. F.; Dutton, P. L. Nature 1994, 368, 425. (b) Bryson, J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X.; O’Neil, K. T.; DeGrado, W. F. Science 1995, 270, 935. (c) Gibney, B. R.; Dutton, P. L. Protein Sci. 1999, 8, 1888-1898. (6) Huffman, D. L.; Rosenblatt, M. M.; Suslick, K. S. J. Am. Chem. Soc. 1998, 120, 6183. (7) Dickerson, R. E.; Geis, I. Hemoglobin: Structure, Function, EVolution and Pathology; Benjamin/Cummings: Menlo Park, CA, 1983. (8) FeIII(copro): Porphyrin Products. His: Sigma. Water: deionized and purified through a Corning MP-1 Megapure system. pH 7 phosphate buffers from reagent grade salts. Peptides: Applied Biosystems 430A peptide synthesizer with HPLC purifications and FAB-MS characterization. (9) (a) Collman, J. P.; Brauman, J. I.; Doxsee, K. M.; Halbert, T. R.; Hayes, S. E.; Suslick, K. S. J. Am. Chem. Soc. 1978, 100, 2761. (b) Suslick, K. S.; Fox, M. M.; Reinert, T. J. J. Am. Chem. Soc. 1984, 106, 4522. (10) O’Neil, K. T.; DeGrado, W. F. Science 1990, 250, 646. (11) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem. 1985, 37, 1. (12) Peptides 4-7 were originally designed in hopes of creating 1:1 complexes with a helix-turn-helix motif, with potential coordination of two histidines. 5418 Inorg. Chem. 2000, 39, 5418-5419