Receptor-induced switch in site-site cooperativity during iron release by transferrin.

Iron removal by PPi from the N- and C-terminal binding sites of both free and receptor-complexed transferrin, when the partner site remains occupied with kinetically inert Co(III), has been studied at pH 7.4 and 5.6, at 25 degrees C. At extracellular pH, 7.4, the C-terminal site of free mixed-metal proteins is slightly more labile than its N-terminal counterpart in releasing iron to 0.05 M PPi. The rate and extent of iron removal are retarded from both sites when transferrins are receptor-bound. At endosomal pH, 5.6, the two sites exhibit greater kinetic heterogeneity in iron release to 0.005 M PPi. The N-terminal site is 6 times more facile in relinquishing iron than the C-terminal site when mixed-metal transferrins are free. However, the two sites are affected oppositely upon binding to the receptor. Iron release from the C-terminal site of receptor-complexed CoN-transferrin-FeC is 4 times faster than that from receptor-free protein. In contrast, iron removal from the N-terminal site of receptor-complexed FeN-transferrin-CoC is slowed by a factor of 2 compared to that from free protein. These results help explain our previous observation of a receptor-induced switch in site lability during iron removal from diferric transferrin at pH 5.6 (Bali & Aisen, 1991). Site-site cooperative interactions between the two sites of doubly-occupied transferrin during iron release are altered upon binding to receptor at pH 5.6. Iron in the otherwise weaker binding site of the N-terminal lobe is stabilized, while iron in the relatively stable binding site of the C-terminal lobe is labilized.     

Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites

Iron release to PPi from N- and C-terminal monoferric transferrins and their complexes with transferrin receptor has been studied at pH 7.4 and 5.6 in 0.05 M HEPES or MES/0.1 M NaCl/0.01 M CHAPS at 25 degrees C. The two sites exhibit kinetic heterogeneity in releasing iron. The N-terminal form is slightly less labile than its C-terminal counterpart at pH 7.4, but much more facile in releasing iron at pH 5.6. At pH 7.4, iron removal by 0.05 M pyrophosphate from each form of monoferric transferrin complexed to the receptor is considerably slower than from the corresponding free monoferric transferrin. However, at pH 5.6, complexation of transferrin to its receptor affects the two forms differently. The rate of iron release to 0.005 M pyrophosphate by the N-terminal species is substantially the same whether transferrin is free or bound to the receptor. In contrast, the C-terminal form releases iron much faster when complexed to the receptor than when free. Urea/PAGE analysis of iron removal from free and receptor-complexed diferric transferrin at pH 5.6 reveals that its C-terminal site is also more labile in the complex, but its N-terminal site is more labile in free diferric transferrin. Thus, the newly discovered role of transferrin receptor in modulating iron release from transferrin predominantly involves the C-terminal site. This observation helps explain the prevalence of circulating N-terminal monoferric transferrin in the human circulation.     

Evidence for functional differences between the two sites of rabbit transferrin: effects of serum and carbon dioxide

A recently developed technique combining urea gel electrophoresis with Western immunoblotting has been modified for assessing the relative ability of each iron binding site of rabbit transferrin in delivering iron to rabbit reticulocytes. The two sites can be made to release iron at the same or differing rates, depending on the experimental conditions. In Hanks' balanced salts solution in an atmosphere of room air or 5% CO2, the acid-labile site in the N-terminal lobe of the protein was found to be 1.4- and 2.9-times more effective than its acid-stable counterpart in providing iron to reticulocytes after 90 min incubation. Both sequential and simultaneous release of iron from the two sites was observed, but sequential release was initiated only from the N-terminal site. The same site also proved to be a better iron donor by a factor of 2 when incubations were conducted in Hanks' medium enriched with 20% serum in 5% CO2. Only in 20% serum in air were the two sites found to be equivalent iron suppliers to reticulocytes. In the cases studied, an atmosphere of 5% CO2 increased 2-fold the effectiveness of iron donation by the acid-labile site to reticulocytes, while the presence of 20% serum enhanced the iron-donating ability of the acid-stable C-terminal site. Thus, the transferrin-reticulocyte interaction is sensitive to environmental variables, and such sensitivity may help account for apparent discrepancies in previous studies of the relative iron-donating abilities of the two sites of transferrin.     

The iron-binding properties of hen ovotransferrin.

1. The distribution of iron between the two iron-binding sites in partially saturated ovotransferrin was studied by labelling with 55Fe and 59Fe and by gel electrophoresis in a urea-containing buffer. 2. When iron is added in the form of chelate complexes at alkaline pH, binding occurs preferentially at the N-terminal binding site. In acid, binding occurs preferentially at the C-terminal site. 3. When simple iron donors (ferric and ferrous salts) are used the metal is distributed at random between the binding sites, as judged by the gel-electrophoresis method. The double-isotope method shows a preference of ferrous salts for the N-terminal site. 4. Quantitative treatment of the results of double-isotope labelling suggests that in the binding of iron to ovotransferrin at alkaline pH co-operative interactions between the sites occur. These interactions are apparently absent in the displacement of copper and in the binding of iron at acid pH.     

Amonabactin-mediated iron acquisition from transferrin and lactoferrin by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> : direct measurement of individual microscopic rate constants

 The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe. The ability of A. hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments. The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis. The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored. Addition of transferrin or lactoferrin promoted bacterial growth. Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing. Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube. Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin. The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access. Taken together, these results support an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin. The implications of these findings for an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed. Received: 8 August 1999 / Accepted: 22 October 1999     

Crystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein.

The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe(3+)-binding protein (hFbp) has been determined to 1.75 A resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21 degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K(d) of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe(3+), while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe(3+) ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe(3+)-binding proteins for Fe(3+) (nFbp K'(eff) = 2.4 x 10(18) M(-1)). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.     

The effect of salts on the kinetics of iron release from N-terminal and C-terminal monoferrictransferrins

The kinetics of iron release from N-terminal and C-terminal monoferric human transferrins has been studied using EDTA as the accepting chelate. In the absence of added salts iron release from the N-terminal site is more facile but the relative lability can be reversed by the addition of NaClO₄, NaCl and LiCl. The results indicate that both anions and cations can affect the lability of the two sites. Since the relative lability of the two monoferrictransferrins is affected by fairly moderate concentrations of NaCl and NaClO₄ we suggest that the ionic composition serum may play an important role in determining the observed distribution of iron among the sites. A new method for the preparation of N-terminal monoferrictransferrin is described.     

Fluorescence and kinetic properties of Ru(III) (NH3)5 modified transferrin

Diferric transferrin was modified using aquopentaammine ruthenium(II), a reagent for surface-accessible uncoordinated histidines. Introduction of the cationic Ru(III) (NH3)3 + 5 group on the imidazole of only 5.5 of the 17 uncoordinated histidines enhances the rates of pyrophosphate-assisted iron removal from the N-terminal and C-terminal binding sites by 16- and 2-fold, respectively. This differential effect on the kinetics of the two sites may partially explain why in the native protein the N-terminal site is more labile than the C-terminal site in acidic solutions where histidine residues become positively charged through protonation. The distance between the metal site and nearby uncoordinated histidines was estimated from fluorescence energy transfer measurements using Tb (III) as the donor and pentaammine ruthenium(III)-labeled imidazole of histidine as the acceptor chromophore. A Tsou Chen-Lu statistical analysis of the fluorescence quenching data suggest that two residues in each lobe of the protein are involved in quenching the fluorescence. By using estimates for the index of refraction and the quantum yield and assuming the energy transfer follows parallel first-order kinetics, an upper limit for the donor-acceptor distance of about 1.4 nm was obtained, assuming two uncoordinated histidine residues equidistant from the metal. His-207 and His-242 in the N-terminal lobe of transferrin and His-535 and His-577 in the C-terminal lobe are within this distance, based on information from the lactoferrin crystal structure. It is postulated that His-207 in the N-terminal lobe and His-535 in the C-terminal lobe are the uncoordinated residues that, when protonated or modified with Ru(III) (NH3)3 + 5, lead to accelerated loss of iron from the two binding sites of the protein.     

Kinetics of pyrophosphate induced iron release from diferric ovotransferrin

The kinetics of pyrophosphate-induced iron release from diferric ovotransferrin were studied spectrophotometrically at 37 degrees C in 0.1 M HEPES, pH 7.0. At high pyrophosphate concentrations, the kinetics are biphasic, indicating that the rates of iron release from the two, presumably noninteracting iron-binding sites of ovotransferrin are different. The pseudo-first-order rate constants for iron release from both the fast and slow sites exhibit a hyperbolic dependence on pyrophosphate concentrations. The data suggest that pyrophosphate forms complexes with the two iron-binding sites of ovotransferrin prior to iron removal. The stability constants of the complex formed with the fast site (Keqf) and slow site (Keqs) are 8.3 M-1 and 40.4 M-1, respectively. The first-order rate constants for the dissociation of ferric-pyrophosphate from the fast site (k2f) and the slow site (k2s) are 0.062 and 0.0044 min-1, respectively. Results from urea gel electrophoresis studies suggest that iron is released at a much faster rate from the N-terminal binding site of ovotransferrin. At high pyrophosphate concentration, only C-monoferric-ovotransferrin is detected during the course of iron release. At low pyrophosphate concentration, however, a detectable amount of N-monoferric-ovotransferrin is accumulated. This result is consistent with the kinetic finding that the site with a higher k2 (0.062 min-1) has a lower affinity toward pyrophosphate (Keq = 8.3 M-1) whereas the site with a lower k2 (0.0044 min-1) has a higher affinity for pyrophosphate (Keq = 40.4 M-1).     

The effects of salts and amino group modification on the iron binding domains of transferrin

The origins of the effects of salts on the properties of the iron binding sites of transferrin have been investigated. The chaotropically distinct salts NaCl and NaClO4 each induce characteristic changes in the EPR lineshapes of the N- and C-terminal Fe3+ binding domains, respectively. To a good approximation the perturbed EPR spectrum of diferric transferrin in the presence of salts is the sum of the EPR spectra of the N- and C-terminal monoferric proteins. Acetylation of amino groups causes spectral and kinetic changes in the protein similar to those induced by NaClO4. Thus, both acetylation and NaClO4 cause a loss of structure in the g' = 4.3 EPR signal of the N-terminal domain, and both retard iron removal from this domain. In contrast, iron removal from the C-terminal domain is accelerated by acetylation or the presence of NaClO4. These observations are ascribed to charge effects of lysine residues which are probably in the vicinity of the iron binding sites.     

Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine

We have investigated the effect of increasing concentrations of methylamine (5, 10, and 25 mM) on the removal of iron from the two iron-binding sites of transferrin during endocytosis by human erythroleukemia (K562) cells. The molecular forms of transferrin released from the cells were analyzed by polyacrylamide gel electrophoresis in 6 M urea. Endocytosis of diferric transferrin was efficient since greater than 10% of surface-bound protein escaped endocytosis and was released in the diferric form. Although transferrin exocytosed from control cells had been depleted of 80% of its iron and contained 65-70% apotransferrin, iron-bearing species were also released (15% C-terminal monoferric; 10% N-terminal; 10% diferric). The ratio of the two monoferric species (C/N) was 1.32 +/- 0.12 (mean +/- SD; n = 4), suggesting that iron in the N-terminal site was more accessible to cells. In the presence of methylamine there was a concentration-dependent increase in the proportion of diferric transferrin release (less than 80% at 25 mM) and a concomitant decrease in apotransferrin. Small amounts of the iron-depleted species, especially apotransferrin, appeared before diferric transferrin, suggesting that these were preferentially released from the cells. The discrepancy between the proportions of the monoferric transferrin species noted with control cells was enhanced at all concentrations of methylamine, most markedly at 10 mM when the C/N ratio was 2.4. The N-terminal site of transferrin loses its iron at a higher pH than the C-terminal site, and so by progressively perturbing the pH of the endocytic vesicle we have increased the difference between the two sites observed with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Terephthalamide-containing ligands: fast removal of iron from transferrin

The mechanism and effectiveness of iron removal from transferrin by three series of new potential therapeutic iron sequestering agents have been analyzed with regard to the structures of the chelators. All compounds are hexadentate ligands composed of a systematically varied combination of methyl-3,2-hydroxypyridinone (Me-3,2-HOPO) and 2,3-dihydroxyterephthalamide (TAM) binding units linked to a polyamine scaffold through amide linkers; each series is based on a specific backbone: tris(2-aminoethyl)amine, spermidine, or 5-LIO(TAM), where 5-LIO is 2-(2-aminoethoxy)ethylamine. Rates of iron removal from transferrin were determined spectrophotometrically for the ten ligands, which all efficiently acquire ferric ion from diferric transferrin with a hyperbolic dependence on ligand concentration (saturation kinetics). The effect of the two iron-binding subunits Me-3,2-HOPO and TAM and of the scaffold structures on iron removal ability is discussed. At the low concentrations corresponding to therapeutic dose, TAM-containing ligands exhibit the fastest rates of iron removal, which correlates with their high affinity for ferric ion and suggests the insertion of such binding units into future therapeutic chelating agents. In addition, urea polyacrylamide gel electrophoresis was used to measure the individual microscopic rates of iron removal from the three iron-bound transferrin species (diferric transferrin, N-terminal monoferric transferrin, C-terminal monoferric transferrin) by the representative chelators 5-LIO(Me-3,2-HOPO)(2)(TAM) and 5-LIO(TAMmeg)(2)(TAM), where TAMmeg is 2,3-dihydroxy-1-(methoxyethylcarbamoyl)terephthalamide. Both ligands show preferential removal from the C-terminal site of the iron-binding protein. However, cooperative effects between the two binding sites differ with the chelator. Replacement of hydroxypyridinone moieties by terephthalamide groups renders the N-terminal site more accessible to the ligand and may represent an advantage for iron chelation therapy.     

Isolation of alpha-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated alpha-subunits and beta-subunits bound to the mitochondrial membrane.

The Makey & Seal [(1976) Biochim. Biophys. Acta 453, 250--256] method of polyacrylamide-gel electrophoresis in buffer containing 6 M-urea was used to determine the distribution of iron between the N-terminal and C-terminal iron-binding sites of transferrin in human serum. In fresh serum the two sites are unequally occupied; there is preferential occupation of the N-terminal site. On incubation of the serum at 37 degrees C the preference of iron for the N-terminal site becomes more marked. On storage of serum at -15 degrees C the iron distribution changes so that there is a marked preference for the C-terminal site. Dialysis of serum against buffer at pH 7.4 also causes iron to be bound much more strongly by the C-terminal than by the N-terminal site. The original preference for the N-terminal site can be resroted to the dialysed serum by addition of the diffusible fraction.     

The carboxy-terminal calcium binding sites of calmodulin control calmodulin's switch from an activator to an inhibitor of RYR1

Calcium and calmodulin both regulate the skeletal muscle calcium release channel, also known as the ryanodine receptor, RYR1. Ca(2+)-free calmodulin (apocalmodulin) activates and Ca(2+)-calmodulin inhibits the ryanodine receptor. The conversion of calmodulin from an activator to an inhibitor is due to Ca(2+) binding to calmodulin. We have previously shown that the binding sites for apocalmodulin and Ca(2+)-calmodulin on RYR1 are overlapping with the Ca(2+)-calmodulin site located slightly N-terminal to the apocalmodulin binding site. We now show that mutations of the calcium binding sites in either the N-terminal or the C-terminal lobes of calmodulin decrease the affinity of calmodulin for the ryanodine receptor, suggesting that both lobes interact with RYR1. Mutation of the two C-terminal Ca(2+) binding sites of calmodulin destroys calmodulin's ability to inhibit ryanodine receptor activity at high calcium concentrations. The mutated calmodulin, however, can still bind to RYR1 at both nanomolar and micromolar Ca(2+) concentrations. Mutating the two N-terminal calcium binding sites of calmodulin does not significantly alter calmodulin's ability to inhibit ryanodine receptor activity. These data suggest that calcium binding to the two C-terminal calcium binding sites within calmodulin is responsible for the switching of calmodulin from an activator to an inhibitor of the ryanodine receptor.     

The influence of pH on the equilibrium distribution of iron between the metal-binding sites of human transferrin.

The dependence of the metal-binding properties of transferrin on pH in the pH 6--9 range was investigated by urea/polyacrylamide-gel electrophoresis. Equations are presented for calculating the relative values of the four conditional site constants for the stepwise binding of iron to the two sites of transferrin and for calculating the equilibrium distribution of the protein among the four principal forms, apotransferrin, the C-terminal and N-terminal monoferric transferrins and diferric transferrin. The relative affinity of iron for the two sites and the co-operativity of iron-binding follow characteristic "pH titration' curves. A mathematical model that can account for the former behaviour is presented. In both cases the metal-binding sites are affected by the ionization of functional groups with apparent pKa values near physiological pH approx. 7.4. There is strong positive co-operatively in the release of protons from these groups. The results indicate that pH must be accurately controlled in studies of the differential properties of the two sites of the transferrin molecule.     

Iron-binding fragments from the carboxyl-terminal region of hen ovotransferrin.

1. When iron-saturated hen ovotransferrin was treated with subtilisin the N-terminal half was digested at a faster rate than the C-terminal half, allowing the latter to be isolated as a single-chain fragment of mol.wt 35000. 2. In mildly acid conditions iron-ovotransferrin loses iron preferentially from its N-terminal binding site. Trypsin digestion of the resulting monoferric ovotransferrin also gave rise to a C-terminal fragment. 3. Comparison of the N-terminal fragment with the C-terminal fragments shows differences in composition, peptide 'maps', CNBr-cleavage patterns and antigenic structures. The C-terminal fragments carry the carbohydrate group of ovotransferrin. 4. Both N-terminal and C-terminal fragments donate their bound iron to rabbit reticulocytes.     

Effect of synthetic carrier ampholytes on saturation of human serum transferrin.

We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds.     

Evidence for the functional equivalence of the iron-binding sites of rat transferrin

The role of the two iron-binding sites of rat transferrin in the exchange of iron with cells has been assessed using urea polyacrylamide gel electrophoresis to separate and quantitate the four possible molecular species of transferrin generated during the incubation of ¹²⁵I-labelled transferrin with rat reticulocytes and hepatocytes. Addition of diferric transferrin to reticulocytes led directly to the appearance of apotransferrin together with small and comparable amounts of the two monoferric transferrins. After 2 h 44.8% of the iron had been removed by the cells, and of the iron-depleted transferrin 71.8% was apotransferrin, the remainder being monoferric transferrin, 16.1% with N-terminal iron and 12.1% with C-terminal iron. A similar pattern emerged with hepatocytes, but the rate of iron removal was slower and the proportion of apotransferrin generated was lower. After 4 h 10.9% of the iron had been removed from the transferrin and the distribution of the iron-depleted protein was: apotransferrin 26.9% and monoferric (N-terminal) 39.2%, (C-terminal) 33.9%. The appearance of apotransferrin during each incubation and the generation of both monoferric transferrins suggest that both cell types are able to remove iron from differic transferrin in pairwise fashion and that they do not appreciably distinguish between the two iron-binding sites of the protein. Release of iron from hepatocytes to apotransferrin lead to the appearance of both monferric species and then to increasing amounts of diferric transferrin. The process of iron release did not seem to distinguish between the vacant iron-binding sites of transferrin.     

Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin.

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change to the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.     

Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin

A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.