METAL-TYROSYL COORDINATION IN TRANSFERRIN. 2. DIFFERENCE ULTRAVIOLET SPECTROSCOPY OF DI-, TRI-, AND TETRAVALENT METAL IONS WITH ETHYLENE-BIS(0-DYDROXYPHENYLGLY-CINE).

In order to probe the metal ion coordination site in the human iron transport protein, transferrin, the complexation of a series of metal ions by the chelate analogue ethylene-bis(o-hydroxyphenylglycine) (EHPG) has been studied by difference uv spectroscopy, in which {Delta}{epsilon} values per coordinated phenol have been determined for the metal complex versus the protonated form of the ligand. With the exception of the.Cu {sup 2+} complex, maxima are observed at 242 nm and 290 nm with a minimum at 269 nm. The {Delta}{epsilon} values at 242 fall into two groups. Complexes of divalent metal ions (Zn{sup 2+}, Cu{sup 2+}, Cd{sup 2+}) have 6£ values ranging from 5000 to 6600 M{sup -1} cm{sup -1} whereas larger {Delta}{epsilon} values are observed for complexes of tri- and tetra- valent metal ions (Th{sup 4+}, Ga{sup 3+}, Fe{sup 3+}, • Ho{sup 3+}, Eu{sup 3+}, Er{sup 3+}, Tb{sup 2+}, VO{sup 2+}), 7400 - 8700 M{sup -1} cm{sup -1}. It is known that the transferrin binding sites contain tyrosyl residues, but there has been considerable debate concerning the precise number of tyrosine groups which bind to specific metal ions. Since it has been the common practice to assume the {Delta}{epsilon} values for coordination by all metal ions are identical, the larger range of {Delta}{epsilon} values actually observed here shows that such an assumption can actually lead to an erroneous tyrosine/metal site ratio. The difference spectra of transferrin and EHPG complexes are very similar, and we have taken the {Delta}{epsilon} values of the EHPG complexes as estimates for the intrinsic {Delta}{epsilon} for coordination of a single tyrosine ligand. The number of tyrosines bound per metal ion is then calculated based on previously reported total {Delta}{epsilon} values of several di(metallo)transferrin complexes. The results show that two tyrosines are coordinated per metal ion for all the transition metals and the smaller lanthanides. Very large metal ions have difficulty fitting into one of the binding sites and the number of coordinated metal ions decreases. This differential ability to coordinate large metal ions lends further support for non-equivalent complexation by the two metal binding regions of transferrin. A model for the Fe{sup 3+} transferrin binding site which is consistent with both these results and previous proton release and chemical modification studies is proposed in which a carbonato, a hydroxo, two tyrosyl, and two histidyl ligands are bound to the ferric ion to form a six-coordinate complex. It is further proposed that the smaller number of protons released upon binding of divalent ions such as Cu{sup 2+} is due to the replacement of the hydroxo group by a water molecule