Synthetic Heme-Peptide Complexes

The “mesomolecular” regime (i.e., from roughly 1000 to 10 000 amu) represents an emerging field that has only recently become synthetically and analytically accessible. We report here the synthesis and characterization of mesomolecular synthetic analogues of heme proteins. While small-molecule studies have provided extremely useful insights into structure-function relationships in heme proteins,1 there often remain significant differences between the properties of the synthetic analogs and the heme proteins themselves. There has been considerable recent interest in peptides that bind metalloporphyrins and the de novo design of artificial heme proteins. Three general classes of complexes have emerged: heme-bound helical bundle peptides;2 peptides covalently attached to the heme periphery;3 and disulfide-dimer peptides coordinated to exchange-inert metalloporphyrins.4 Relatively little is yet known, however, about the factors that influence peptide binding to metalloporphyrins. We have prepared a series of peptides that form 1:2 metalloporphyrinpeptide complexes and examined the effect of their sequence on binding constants, secondary structure, and electrochemical behavior. The utilization of peptides as ligands for metalloporphyrins poses several challenges: viz., control of conformational demands to favor metal coordination, enhancement of water solubility, and optimization of intramolecular interactions. To simplify characterization and interpretation, we have limited our peptides to amphiphilic 15-mers with a restricted repertoire of amino acid residues. We chose this size because this is roughly the minimum necessary to form good R-helices (∼4 turns).5 For comparison, a metalloporphyrin is about 1.0 nm across, i.e., about two helix turns (0.54 nm per turn). The peptides also possess a palindromic sequence symmetry about the central ligating residue (in these cases, the imidazole of a histidine). To delineate the factors that stabilize heme-peptide complexes, we have systematically altered key peptide residues in contact with the heme. The remaining residues of the sequences were chosen to encourage helix formation and good solubility. For strong intrinsic helix-forming ability,6 Aib,7 Ala, Leu, Lys, and Nva were used. To probe hydrophobic interactions, Phe, Leu, Nva, Ala, and Ser were examined. Peptide solubility was provided by Lys residues in every third or fourth position. The sequence termini were capped to enhance helix dipole formation.8 A list of the peptide sequences is shown in Table 1. It should be noted that the location of His in the middle tends to destabilize helix formation by the free peptide, since His is a helix breaker. Coproporphyrin-Iatoiron(III) chloride, FeIII(copro)(Cl), (Figure 1) was chosen as the metalloporphyrin for its excellent aqueous solubility, nonaggregation, and high symmetry. Binding constants (Table 1) of the peptides with FeIII(copro) were determined by spectrophotometric analysis of titration data using standard methods.10 Surprisingly, the heme-peptide binding constants span a range (1) Collman, J. P. Inorg. Chem. 1997, 36, 5145. (b) Traylor, T. G. Pure Appl. Chem. 1991, 63, 265. (c) Van Deusen-Jeffries, S.; Suslick, K. S. ComprehensiVe Supramolecular Chemistry; Lehn, J. M., Ed.; Elsevier Publishers: Oxford, 1996; Vol. 5, pp 141-170. (2) 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 and references therein. (3) Arnold, P. A.; Benson, D. R.; Brink, D. J.; Hendrich, M. P.; Jas, G. S.; Kennedy, M. L.; Petasis, D. T.; Wang, M. Inorg. Chem. 1997, 36, 5306 and references therein. (b) Nastri, F.; Lombardi, A.; Morelli, G.; Maglio, O.; D’Auria, C. P.; Pavone, V. Chem. Eur. J. 1997, 3, 340. (c) Karpishin, T. B.; Vannelli, T. A.; Glover, K. J. J. Am. Chem. Soc. 1997, 119, 9063. (4) Arnold, P. A.; Shelton, W. R.; Benson, D. R. J. Am. Chem. Soc. 1997, 119, 3181. (b) Sakamoto, S.; Sakurai, S.; Ueno, A.; Mihara, H. Chem. Commun. 1997, 1221. (5) Padmanabhan, S.; Marqusee, S.; Ridgeway, T.; Laue, T. M.; Baldwin, R. L. Nature 1990, 344, 268. (6) Shoemaker, K. R.; Kim, P. S.; York, E. J.; Stewart, J. M.; Baldwin, R. L. Nature 1987, 326, 563. (b) O’Neil, K. T.; DeGrado, W. F. Science 1990, 250, 646. (c) Pingchiang, C. L.; Sherman, J. C.; Chen, A.; Kallenbach, N. R. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 5317. (7) Basu, G.; Baghi, K.; Kuki, A. Biopolymers 1991, 31, 1763. (8) Hol, W. G. J.; vanDuijnen, P. T.; Berendsen, H. J. C. Nature 1978, 273, 443. (9) Chen, Y. H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350. (10) Isosbestic behavior (Figure 1) showed that there were only two absorbing species in solution, and spectrophotometric data analysis confirmed 2:1 peptide-metalloporphyrin complex formation for all cases. Visible spectra confirmed bis-imidazole complexation, and EPR spectra were consistent with the expected low spin (S ) /2) iron(III) center.10b (b) Huffman, D. L. Ph.D. Thesis, University of Illinois at UrbanasChampaign, 1994. Also see the Supporting Information. Figure 1. Spectrophotometric titration of coproporphyrin-I-ato iron(III) with peptide AA-A.