1H NMR studies of porcine uteroferrin. Magnetic interactions and active site structure.

Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean

“…The chemical shifts of the two pairs of signals a, e and b, bh are almost coincident with those of the resonances assigned to the CβH and CδH protons, respectively, of the tyrosine bound to Fe(III) in Uf [13][14][15]. This striking similarity suggests that the same assignments also apply for the present di-iron derivative of KBPAP.…”

“…Therefore, on the basis of the X-ray structure [10], peaks a and e and peaks b and bh can be assigned to the CβH and CδH protons, respectively, of Tyr-167. The remarkable broadening of b and bh as compared with the corresponding peaks in Uf (c and d), which are well resolved [13], may be tentatively attributed, besides to the effects of Curie relaxation, to an intermediate exchange of the two tyrosine CδH protons on the NMR time scale due to ring flipping [13,15]. This cannot be verified by decreasing the temperature, because of the dramatic increase in efficiency of Curie relaxation and the consequent general broadening of the spectral features.…”

“…Their spectral parameters are listed in Table 1. Since hyperfine shifts of protons in the surroundings of both Fe(III) and Fe(II) centres are mainly contact in origin [14][15][16][17], these resonances most probably arise from metal-co-ordinating residues. Incidentally, we note that the present protein derivative is the largest non-haem paramagnetic iron protein for which resolved hyperfine-shifted "H NMR signals have been obtained [16].…”

“…Although Uf and KBPAP are scarcely sequence related (about 20 % identity), sequence-alignment studies suggest that the metal-binding residues should be conserved [9]. Moreover, the recently solved X-ray structure of the plant enzyme [10,11], which is the only three-dimensional structure of PAPs available to date, shows that striking analogies exist between the nature and arrangement of the metal-binding residues of the bimetallic core of the protein and those proposed for the mammalian PAPs on the basis of "H NMR measurements [12][13][14][15]. In order to probe for these similarities on spectroscopic grounds, we have hereby exploited the hyperfine-shifted "H NMR resonances [16,17] as a sort of spectral fingerprints of the active site.…”

“…been replaced by an Fe(II) ion [4,5], thus reproducing in the plant enzyme the di-iron core of the mammalian species. In the comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian enzymes, which were previously extensively assigned [12][13][14][15], we profit from the known three-dimensional structure of the former species to verify assignments and probe for conserved features of the metal-binding domains. Spectral comparison was also extended to the Fe(III)Co(II) derivatives [5,12].…”