Electron spin echo envelope modulation evidence for carbonate binding to iron(III) and copper(II) transferrin and lactoferrin.

Iron binding to transferrin and lactoferrin requires a synergistic anion, which is carbonate in vivo. The anion is thought to play a key role in iron binding and release. To understand better the iron-carbonate interaction, experiments were performed with iron(III) and copper(II) complexes of human milk lactoferrin and serum transferrin with carbon-13-labeled carbonate. Modulation frequencies were present in the Fourier transforms of two-pulse and three-pulse electron spin echo envelope modulation data for the Fe(III) and Cu(II) complexes, consistent with binding of carbonate to both metals. The metal-13C interaction was similar for the lactoferrin and transferrin complexes. Spin coupling to the nitrogen of a coordinated histidine imidazole was observed for both metals. Both the metal-nitrogen and the metal-carbon spin coupling constants were about a factor of 5 smaller for the iron complexes than for the copper complexes, which indicated substantial similarity in the metal-carbonate and metal-imidazole binding for the two metals.

were about a factor of 5 smaller for the iron complexes than for the copper complexes, which indicated substantial similarity in the metal-carbonate and metal-imidazole binding for the two metals.
The transferrins are a family of iron-binding proteins that include transferrin (Tf)' and lactoferrin (Lf). These proteins are found in the physiological fluids of a wide variety of vertebrates (1,2). The role of transferrin in iron transport has been studied extensively (1, 2). The roles of lactoferrin in regulation of myelopoiesis, immune response, microbicidal and bacteriostatic activity, and the anemia of inflammation have been reported recently (3-8). X-ray crystal structures have confirmed that both proteins have two metal-binding sites that are spatially separated (9, 10). Occupancy of these sites requires binding of an anion (1, 2). The proteins bind iron with extremely high equilibrium constants but readily release iron as needed by cells. The anion is thought to play a crucial role in iron binding and release, so it is important to elucidate the interaction with the iron. The protein provides four ligands for the metal, leaving two open sites for coordination of the anion and/or water (9, 10). A variety of spectroscopic techniques has shown that the anion-binding site is in close proximity to the metal-binding site (1,11 2615 G is near the low field extreme of the CW EPR  (Fig. 26) but not for Cu. Tf. CO3 (Fig. 2a). A peak at 6.9 MHz also was present in the twopulse data. On the basis of the similarity with the 13C modulation frequency observed at 2615 G, the peak at 7.1 MHz is assigned to a nearly parallel orientation of molecules with copper ml = i/2. The 7.0-MHz peak for Cu.Lf. [13C]C03 at 2800 G is assigned similarly.  Analysis of the observed 13C modulation frequencies with Equation 1 gave the copper-13C coupling constants shown in Table I. The observed values of 6.4-8.4 MHz imply an isotropic contribution of 7.4 f 0.5 MHz. The smallest anisotropic contribution consistent with these values is 0.7 MH~,~which corresponds to a copper-'3C dipolar interaction at 3.1 A. The ESEEM data were obtained for the magnetic field along the copper z axis, in the copper xy plane, and at an intermediate orientation with respect to the copper axis system. Since the orientation of the copper-13C vector within the copper axis system is not known, the data may not* have sampled the maximum dipolar interaction.
Thus, 3.1 A is an upper limit on the copper-13C distance. The magnitude of the isotropic interaction requires through-bond interaction between the copper and the carbonate carbon. The copper-13C distance is consistent with direct coordination of the carbonate to the copper.
The cosine Fourier transform of the three-pulse ESEEM for Fe.Tf.COB at a magnetic field of 1580 G is shown in Fig.  3a. When [%]carbonate was used to prepare the complex, an additional peak at 3.2 MHz (Fig. 3b) was observed. Similarly, a peak at 3.2 MHz was observed for Fe. Lf. [13C]C03 ( Fig. 3c) but not for FebLf.C03.
The frequencies of these peaks indicate (Equation 3) that the iron-13C coupling was about 1.5 MHz. This value is approximately one-fifth of the value observed for the analogous copper complex. If a single unpaired electron dominates a metal-nuclear spin-spin interaction and the bonding remains approximately constant, the coupling constant is expected to vary as l/n where n is the number of unpaired electrons on the metal (21, 22). Since Cu(I1) has n = 1 and Fe(II1) has n = 5, the observed decrease in the coupling constant by a factor of 5 indicates that the bonding between the metal and the carbonate is similar for the two metals. Nitrogen Modulation-The nitrogen modulation frequencies for Cu.Tf.CO, and Cu. Lf.CO, at a magnetic field of 3200 G have been reported previously (15). For the three magnetic fields examined in this study, the nitrogen frequencies in the range of 0.7-1.6 MHz were approximately constant ( Table I). As noted in the analysis of ESEEM of other copper complexes of histidine imidazoles, these frequencies are almost purely quadrupolar, which occurs at "near cancellation," i.e. when the nuclear Zeeman frequency is about half the isotropic coupling (13, 23).
The frequency of the peak at 3.5-4.1 MHz depends on the electron-nitrogen coupling as well as the nitrogen nuclear ESEEM of Iron and Copper(U) Transferrin and Lactoferrin 7141 frequency and the quadrupole parameters (Equation 4). The peak at 4.05-4.10 MHz at 3185 G is from molecules with the magnetic field in the perpendicular plane of the copper. At 2615 G the 3.5-MHz peak is due to the parallel orientation of molecules with copper ml = %. At 2800-2810 G the nitrogen frequency of 3.5 MHz is assigned to a nearly parallel orientation of molecules with copper ml = i/z on the basis of the similarity with the frequency observed at 2615 G, and the 3.7and 3.9-MHz frequencies are assigned to intermediate orien- tations of molecules with copper ml = %. The orientation dependence of the copper-nitrogen coupling is relatively small (Table I). This is consistent with assignment of the coupling to interaction with the distant nitrogen of the coordinated histidine imidazole since the long distance would result in a small dipolar contribution to the interaction. The three nitrogen frequencies at 0.70-0.75, 0.80, and 1.57 in the ESEEM data for Fe. Tf. CO, and Fe. Lf. CO, are within experimental uncertainty of the three frequencies observed for the analogous copper complexes. By analogy, these frequencies are assigned as predominantly quadrupolar, arising from interaction with the distant nitrogen of the coordinated histidine imidazole. Apparently the conditions for near cancellation are also satisfied for the iron complex. As discussed under "Methods," near cancellation for the middle Kramer's doublet of Fe. Tf. CO, requires that the nuclear Zeeman frequency be approximately equal to the nuclear hyperfine coupling. At 1580 G, the nitrogen nuclear Zeeman frequency is 0.49 MHz so oN is -0.5 MHz. Alternatively, the nitrogen hyperfine coupling can be estimated from the observed frequency of 2.3 MHz and Equation 5, which gave oN -0.35 MHz. Together, the two approaches suggest oN -0.4 MHz. This value also is about one-fifth of the value observed for the copper complex, which indicates that the metal-imidazole binding is similar for the two metals. in the quadrupolar nitrogen modulation frequencies for the iron and copper complexes is consistent with interaction with the distant nitrogen of a coordinated histidine imidazole for both metals. The ironnitrogen and iron-carbon coupling constants were about a factor of 5 smaller than the analogous copper-nuclear coupling constants, which implies substantial similarity in the metalligand bonding for the two metals. This spectroscopic study provides strong evidence that the carbonate anion binds di-rectly to the metal in iron and copper complexes of transferrin and lactoferrin.
The spectroscopic data indicate substantial similarity between the metal-anion-binding sites for transferrin and lactoferrin. X-ray crystal structures have shown that the overall topologies also are very similar for the two proteins and that the metal-anion-binding sites occur at the interface between two dissimilar domains (9, 10). Iron release is known to occur with a large change in protein conformation.
Whether this change is triggered by events at the metal-anion-binding site or at the protein surface remains to be determined.