Structure of iron saturated C-lobe of bovine lactoferrin at pH 6.8 indicates a weakening of iron coordination

The bilobal lactoferrin is an approximately 76 kDa glycoprotein. It sequesters two Fe3+ ions together with two CO32- ions. The C-terminal half (residues, Tyr342-Arg689, C-lobe) of bovine lactoferrin (BLF) (residues Ala1-Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact BLF were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 angstrom resolution and refined to R-cryst and R-free factors of 0.205 and 0.273, respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 angstrom for their C-alpha atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 angstrom(2). The distance of 2.9 angstrom observed for the coordination bond between Fe3+ ion and N-e2 of His595 appeared to be considerably longer than the normally observed values of 1.9-2.2 angstrom. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1-2.3 angstrom. Based on the present structure of iron saturated C-lobe, it may be stated that His595 is the first residue to dissociate from ferric ion when the pH is lowered. (C) 2016 Wiley Periodicals, Inc.