1H NMR relaxometric characterization of bovine lactoferrin.
Lactoferrin (Lf) is a mammalian iron binding protein present in external secretions and in polymorphonuclear leukocytes. Its role in host defense mechanisms related to the non-immune defense system has been definitively established. Lf has two identical iron-binding sites, far from each other (44.3 A) and magnetically non-interacting. Fe(III) ions are six-coordinated, with four donor atoms provided by protein sidechains (two Tyr, one His, one Asp) and two oxygen atoms from a bridged HCO(3)(-). This set of ligands provides an ideal coordination scheme for stable and reversible iron binding. Nuclear magnetic relaxation dispersion (NMRD) profiles of Lf are consistent with a closest distance for a single water hydrogen atom of 3.1 A. By looking at the X-ray structure of Lf (PDB ID code: 1BLF) we can locate two water oxygens at 3.95 and 4.27 A from each Fe(III), respectively. Temperature dependence data suggest that an important contribution to the overall paramagnetic contribution to the solvent water relaxation rate arises from one or more second sphere water molecules in slow exchange with the bulk. A decreasing value of the exchange rate is obtained, ranging from 1.2 to 0.7 micros in the observed temperature range (25-65 degrees C), with an activation enthalpy of 7.3+/-0.8 kJ mol(-1). The low exchange rate obtained from NMRD data can be explained by the observation that both water molecules are bound to several polar groups of the protein backbone and side chains. By increasing the pH from 6.5 to 12 two distinct titrations are observed, consistent with sequential removal of both water molecules.
Dipartimento di Biologia Strutturale e Funzionale, Università dell'Insubria, Via Alberto da Giussano 12, I-21052 Busto Arsizio (VA), Italy. mauro.fasano@uninsubria.it
Mauro
M
Fasano
Gabriella
G
Fanali
Fabio
F
Polticelli
Paolo
P
Ascenzi
Giovanni
G
Antonini
D000818:Animals; D002417:Cattle; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007781:Lactoferrin / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D012997:Solvents / Q000737:chemistry; D014867:Water / Q000737:chemistry
D012997:Solvents; D014867:Water; D007501:Iron; D007781:Lactoferrin