Inorg. Chem. zyxwvuts 1994,33, zyxwvu 5819-5824 5819 Polarographic and Spectrophotometric Investigation of Iron(II1) Complexation to zy 3,4-Dihydroxyphenylalanine-Containing Peptides and Proteins from Mytilus edulis Steven W. Taylor,* George W. Luther 111, and J. Herbert Waite College of Marine Studies and Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 Received February 3, zyxwvutsr 1994@ The iron(II1) binding properties of Mytilis edulis foot protein 1 (Mefpl), its component peptides hexapeptide A (A~KZP~T~Y~~; Y5 zyxwvutsr = 3,4-dihydroxyphenylalanine (DOPA)) and decapeptides B to E (AlK2P3S4Y5P6P7TgYgKl~; peptides B to E: P6 = 3-hydroxyproline (3HYP), P7 = 4HYP, Yg = DOPA; peptide B: P3 = 4HYP, Y5 = DOPA; peptide C: P3 = 4HYP; peptide D: Y5 = DOPA), and their synthetic decapeptide analogues (peptide SI: Yg = DOPA; peptide Sz: Y5 = Yg = DOPA) have been investigated by polarography and spectrophotometry. A “chelate scale” was constructed by measuring the reduction potentials of iron(III) complexes with known stability constants and was used to estimate stability constants for the iron(II1) interactions with peptides. A linear relationship was found to exist between the reduction potentials and the pH independent thermodynamic stability constants for iron(II1) complexes spanning 20 orders of magnitude in chelate stability. Spectrophotometric data allowed the stoichiometry of the peptide-iron(II1) interactions to be determined. At neutral pH all peptides favored an intermolecular bis(catecho1ato) coordination mode, through the DOPA nearest the C terminus. Reasons for the extra stability observed in the peptide complexes compared with simple catecholates are offered, with the possibility of hydroxyproline involvement eliminated on the basis of comparisons of behavior of the natural peptides with that of their synthetic analogues. The decapeptides provide good models for iron(II1) coordination by the parent protein. The biological and technological relevance of the iron(1II)-DOPA interactions is discussed. Introduction Mussels are known to accumulate high concentrations of metal ions from seawater. Many of these metals have been detected in their byssal threads at levels which often implicate these holdfasts as candidates for environmental markers of metal pollution. 1-3 Certain mytilid species display a degree of specificity for the type metal ion that is sequestered. Tateda and Koyanagi, for example, found that Septifer virgatus preferentially accumulated manganese from seawater while the common mussel Mytilus edulis sequestered higher concentra- tions of iron! Iron is predominantly found in the adhesive plaques or disks (ca. 1700 pg/g of zyxwvuts dry weight) but also in the threads (ca. 400 pglg of dry weight) of M. edulis byssus.2 Radioactive tracer studies employing iron-59 have shown that the metal accumulates in linear proportion to the seawater concentration, about 30% of that absorbed in the gut being ultimately excreted in the faeces, while 35% is transported by amoebocytes in the hemolymph from the gut to the byssus via the byssus gland complex.’ Polyphenolic proteins derived from a part of this complex, known as the phenol gland, and more recently from the byssal threads and plaques, have been intensively in~estigated.~-’~ A novel characteristic of many of these proteins is the presence of 3,4-dihydroxyphenylalanine (DOPA) in their primary struc- ture, produced by a post- or co-translational hydroxylation of tyr~sine.~J~ Waite5s6 characterized peptides from tryptic digests of the first of these families of proteins, designated M. edulis foot protein 1 (Mefpl). His results suggested that Mefpl consisted predominantly of tandemly repeated hexa- and de- capeptide sequences with extensive hydroxylation of tyrosine to DOPA and also proline to 3- and 4-trans-hydro~yproline~9~ (Figure 1). Filpula et al. and Laursen later confirmed this hypothesis by sequencing the appropriate genomic and cD- The catecholic moiety of DOPA gives the proteins potent complexing power, the catechol siderophores forming some of the strongest complexes with iron(II1) known.14 The chelating potential of DOPA proteins may be important to processes of adhesion both by surface coupling with metals in marine substrata and by aiding in the process of quinone tanning to form an intractable, resistant matrix.15 In this paper we describe the complexation of iron(II1) by both Mefpl and its component peptides which have been isolated from tryptic digests of the parent protein as well as some synthetic decapeptide analogues. Since only submilligram quantities of these peptides were typically available, a sensitive NA’s.12,13 * To whom correspondence should be addressed at the Department of @ Abstract published in Advance ACS Abstracts, November 1, 1994. (1) George, zyxwvutsrqpo S. G.; Pirie, B. J. S.; Coombs, T. L. J. Exp. Mar. Biol. Ecol. (2) Coombs, T. L.; Keller, P. J. Aquat. Toxicol. zyxwvutsrq 1981, 1, 291. (3) Koide, M; Lee, D. S.; Goldberg, E. D. Est. Coast. ShelfSci. 1982, (4) Tateda, Y.; Koyanagi, T. Bull. Jpn. SOC. Fish. 1986, 52, 2019. (5) Waite, J. H. J. Biol. Chem. 1983, 258, 2911. (6) Waite, J. H.; Housley, T. J.; Tanzer, M. L. Biochemistry 1985, 24, (7) Pardo, J.; Gutierrez, E.; SBez, C.; Brito, M.; Burzio, L. 0. Prot. Exp. Chemistry and BiochemistIy. Fax: 302-831 6335. 1976, 23, 71. 15, 679. 5010. Purif. 1990, 1, 147. 0020- 166919411 333-58 19$04.50/0 (8) Rzepecki, L. M.; Chin, SA; Waite, J. H.; Lavin, M. F. Mol. Mar. (9) Marumo, K.; Waite, J. H. Biochim. Biophys. Acta 1986, 872, 98. (10) Rzepecki, L. M.; Hansen, K. M.; Waite, J. H. Biol. Bull. 1992, 183, (11) Benedict, C. V.; Waite, J. H. J. Morphol. 1986, 189, 171. (12) Filpula, D. R.; Lee, S.-M.; Link, R. P.; Strausberg, S. L.; Strausberg, R. L. Biotechnol. Prog. 1990, 6, 171. (13) Laursen, R. A. In Results and Problems zyxw in Cell Differentiation; Case, S. T., Ed.; Springer-Verlag: Berlin, 1992; Vol. 19, Chapter 3. (14) Raymond, K. N.; Muller, G.; Matzanke, B. F. In Topics in Current Chemistry; Boschke, F. L., Ed.; Springer-Verlag: Berlin, 1984; Vol. Biol. Biotech. 1991, 1, 78. 123. 123, pp 50-102. (15) Waite, J. H. Comp. Biochem. Physiol. 1990, 97B, 19. 0 1994 American Chemical Society