SIDEROPHILIN METAL COORDINATION - DIFFERENCE ULTRAVIOLET SPECTROSCOPY OF DIVALENT, TRI-VALENT, AND TETRAVALENT METAL-IONS WITH ETHYLENEBIS[(ORTHO-HYDROXYPHENYL)GLYCINE]

So that the metal ion coordination site in the human iron transport protein, transferrin, could be probed, the complexation of a series of metal ions by the chelate analog ethylenebis[(o-hydroxyphenyl)glycine] (EHPG) was studied by difference UV spectroscopy, in which .DELTA..epsilon. [change in extinction coefficient] values/coordinated phenol were determined for the metal complex vs. the protonated form of the ligand. With the exception of the Cu2+ complex, maxima are observed at 242 and 290 nm, with a minimum at 269 nm. The .DELTA..epsilon. values at 242 nm fall into 2 groups. Complexes of divalent metal ions (Zn2+, Cu2+ and Cd2+) have .DELTA..epsilon. values ranging from 5000-6600 M-1 cm-1, whereas larger .DELTA..epsilon. values are observed for complexes of tri- and tetravalent metal ions (Th4+, Ga3+, Fe3+, Ho3+, Eu3+, Er3+, Tb3+ and VO2+), 7400-8700 M-1 cm-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 that 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 tryosine/metal site ratio. The difference spectra of transferrin and EHPG complexes are very similar, and the .DELTA..epsilon. values of the EHPG complexes were taken as estimates for the intrinsic .DELTA..epsilon. for coordination of a single tyrosine ligand. The number of tyrosines bound/metal ion is then calculated on the basis of previously reported total .DELTA..epsilon. values of several dimetallotransferrin complexes. Two tyrosines are coordinated/metal ion for all the transition metals and the smaller lanthanides. Very large metal ions have difficulty fitting into 1 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 2 metal binding regions of transferrin. A model for the Fe3+ 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, 2 tyrosyl and 2 histidyl ligands are bound to the ferric ion to form a 6-coordinate complex. The smaller number of protons released upon binding of divalent ions, such as Cu2+, may be due to the effective replacement of the hydroxo group by a H2O molecule, due to the lower acidity of the dication aquo complexes.