Iron-binding sites

Figure 5.7 Structures of the alternative conformations of the iron-binding sites in the orthorhombic crystal form of the recombinant N-lobe of human transferrin. Reproduced with permission from MacGillivray et ah, 1998. Copyright (1998), American Chemical Society. 

The iron-binding sites have been characterized by crystallographic studies on several transferrins, and in Figure 5.7 (Plate 7) that of the N-lobe of human lactoferrin is presented. The 3+ charge on the ferric ion is matched by the three anionic ligands Asp-63, Tyr-95 and Tyr-188 (the fourth, His-249, is neutral), while the charge on the carbonate anion is almost matched by the positive charge on Arg-124 and the... 

A recently obtained high resolution structure of two crystal forms of the N-lobe of human serum transferrin (at 0.16 and 0.18 nm resolution) shows disorder at the iron-binding sites (MacGillivray et ah, 1998). Model building and refinement show... 

Several binding sites for Tb3+ or Cd2+ ions have been identified in the interior of the apoferritin protein shell, some of which may be iron-binding sites (Harrison et ai, 1989 Granier et ah, 1998). In HoSF and HoLF, two sites were identified on the inner surface of the B helix at the subunit dimer interface (Figure 6.15, Plate 11) which bind two Cd2+ ions. One involves Glu-57 and Glu-60 as ligands and the other Glu-61 and Glu-64 (Granier et al., 1998). In H-chain ferritins the first pair of Glu-57 and Glu-60 are both replaced by His and only a single Tb3+ is found bound to Glu-61 and Glu-64 (Lawson et al, 1991). 

Under normal physiological conditions, iron is transported in serum by transferrin, an 80 kDa bilobal protein with two almost identical iron-binding sites, one in each half of the molecule. 

Apart from fluorescence, several other methods may be used to obtain time-resolved information. In the case of proteins containing an iron atom, Mossbauer spectroscopy allows the determination, in the iron binding site, of not only root-mean-square shifts of atoms but also the times over which such shifts occur. Detailed investigations of myoglobin have yielded relaxation times on the order of 10 8 Proton NMR spectroscopy can be used to... 

Agar JN, Yuvaniyama P, Jack RF, et al. 2000b. Modular organization and identification of a mononnclear iron-binding site within the NifU protein. J Biol Inorg Chem 5 167-77. 

Fig. 2.12 Examples of non-linear Arrhenius (or Eyring) plots (a) 1u(A oh)7 " ) vs T for the base hydrolysis of trans-Co(en)2ClJ. Curvature may result when k, k2 and A// , not equalling A// in the conjugate-base mechanism (Sec. 4.3.4). Reprinted with permission from C. Blakeley and M. L. Tobe, J. Chem. Soc. Dalton Trans. 1775 (1987). (b) nk vs T for iron removal from C- and N-terminal monoferric transferrin (lower and upper scales respectively). Transferrin contains two iron binding sites = 35 A apart. Either of the two sites, designated C- and N-terminal, can be exclusively labelled by Fe(lll) ions and these may be removed by a strong ligand such as a catechol (see Sec. 4.11). Reprinted with permission from S. A. Kretschmar and K. N. Raymond, J. Amer. Chem. Soc. 108, 6212 (1986). (1986) American Chemical Society.

Multisites in proteins are not uncommon. The removal of metal ions from such centers is likely to be involved. This is in fact illustrated by the iron removal from serotransferrin, see also Sec. 2.6. This protein is bilobal and each lobe contains an iron-binding site. These are 35 A apart and it is believed that direct interaction between the sites is absent. The two Fe s, designated a and b, are different and their removal is biphasic, although not markedly so. 

The degeneracy of the non-chiral complexes can be removed by incorporating chiral centers, usually as resolved amino acids, into the arms at close vicinity to the hydroxamate iron binding sites. Thus, only one of the energetically non-equivalent diastereomers predominates, leading to pure enantiomeric iron(III) complexes with defined hehcity that allows assessing stereospecific recognition by the ferrichrome receptor. 

Detailed pictures of the iron-binding sites in transferrins have been provided by the crystal structures of lactoferrin (Anderson et ai, 1987, 1989 Baker etai, 1987) and serum transferrin (Bailey etal., 1988). Each structure is organized into two lobes of similar structure (the amino- and carboxy-terminal lobes) that exhibit internal sequence homology. Each lobe, in turn, is organized into two domains separated by a cleft (Fig. 3 and 10). The domains have similar folding patterns of the a//3 type. One iron site is present in each lobe, which occupies equivalent positions in the interdomain cleft. The same sets of residues serve as iron ligands to the two sites two tyrosines, one histidine, and one aspartate. Additional extra density completes the octahedral coordination of the iron and presumably corresponds to an anion and/or bound water. The iron sites are buried about 10 A below the protein surface and are inaccessible to solvent. 

Metal storage proteins also form a variety of nitrosyl complexes. Metallothi-onein has been shown to form a low-spin complex with Fe and NO (Kennedy et al., 1993). Ferritin contains multiple iron binding sites capable of forming spectroscopically distinguishable nitrosyl complexes (Lee et al., 1994 LeBrun et al., 1993). 

The molecular weights of rubredoxin from different sources are about 6000. The proteins from D. vulgaris and D. gigas have 37 of their 52 and 54 amino acid residues in common, but only 12 residues identical with rubredoxin from other bacteria. These 12 residues are concerned mainly with the iron-binding sites. 

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