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 A resolution and refined to Rcryst and Rfree 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 A for their Cα atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 A2. The distance of 2.9 A observed for the coordination bond between Fe3+ ion and Ne2 of His595 appeared to be considerably longer than the normally observed values of 1.9–2.2 A. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1–2.3 A. 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. Proteins 2016; 84:591–599. © 2016 Wiley Periodicals, Inc.