Laser-induced lanthanide luminescence as a probe of metal ion-binding sites of human Factor Xa.

7F0 ---- 5D0 excitation spectroscopy of Eu(III) has shown that human Factor Xa has two high affinity lanthanide ion-binding sites. The deuterium isotope effect on the reciprocal lifetime (tau-1) of excited Eu(III) in human Factor Xa has indicated that 2 to 3 water molecules remain on Eu(III) after being complexed by Factor Xa, suggesting that 3-6 ligand atoms are provided by the protein, probably through two or three gamma-carboxyglutamic acids (GLA). F orster -type interlanthanide energy transfer has been utilized to measure the distance between the high affinity metal ion-binding sites of human Factor Xa using Tb(III) as an energy donor and Nd(III), Ho(III), or Er(III) as energy acceptors. Tau-1 values of Tb(III) in the presence of the acceptor ions Nd(III), Ho(III), and Er(III) were 1.90, 1.66, and 1.76 ms-1, respectively, which compared to 1.31 ms-1 in the presence of the nonacceptor ion Gd(III), yield energy transfer efficiencies of 0.29, 0.20, and 0.24, respectively. From these efficiencies and published critical distances (R0) ( Horrocks , W. DeW ., Jr., Rhee , M-J., Snyder, A. P., and Sudnick , D. R. (1980) J. Am. Chem. Soc. 102, 3650-3652), the distance between two high affinity sites is estimated to be 10.7 A. Based on these data, we propose that the two high affinity sites of human Factor Xa consist of two paired GLA residues; GLA-19, GLA-20 and GLA-25, GLA-26 together with one of the remaining single GLA residues for each site.