Aluminum exchange between citrate and human serum transferrin and interaction with transferrin receptor 1

The kinetics and thermodynamics of Al(III) exchange between aluminum citrate (AlL) and human serum transferrin were investigated in the 7.2-8.9 pH range. The C-site of human serum apotransferrin in interaction with bicarbonate removes Al(III) from Al citrate with an exchange equilibrium constant K1 = (2.0 +/- 0.6) x 10(-2); a direct second-order rate constant k1 = 45 +/- 3 M(-1) x s(-1); and a reverse second-order rate constant k(-1) = (2.3 +/- 0.5) x 10(3) M(-1) x s(-1). The newly formed aluminum-protein complex loses a single proton with proton dissociation constant K1a = (15 +/- 3) nM to yield a first kinetic intermediate. This intermediate then undergoes a modification in its conformation followed by two proton losses; first-order rate constant k2 = (4.20 +/- 0.02) x 10(-2) s(-1) to produce a second kinetic intermediate, which in turn undergoes a last slow modification in the conformation to yield the aluminum-loaded transferrin in its final state. This last process rate-controls Al(III) uptake by the N-site of the protein and is independent of the experimental parameters with a constant reciprocal relaxation time tau3(-1) = (6 +/- 1) x 10(-5) x s(-1). The affinities involved in aluminum uptake by serum transferrins are about 10 orders of magnitude lower than those involved in the uptake of iron. The interactions of iron-loaded transferrins with transferrin receptor 1 occur with average dissociation constants of 3 +/- 1 and 5 +/- 1 nM for the only C-site iron-loaded and of 6.0 +/- 0.6 and 7 +/- 0.5 nM for the iron-saturated ST in the absence or presence of CHAPS, respectively. No interaction is detected between receptor 1 and aluminum-saturated or mixed C-site iron-loaded/N-site aluminum-loaded transferrin under the same conditions. The fact that aluminum can be solubilized by serum transferrin in biological fluids does not necessarily imply that its transfer from the blood stream to cytoplasm follows the receptor-mediated pathway of iron transport by transferrins.     

Transferrin, is a mixed chelate-protein ternary complex involved in the mechanism of iron uptake by serum-transferrin in vitro?

Iron uptake by transferrin from triacetohydroxamatoFe(III) (Fe(AHA)3) in the presence of bicarbonate has been investigated between pH 7 and 8.2. The protein transits from the opened apo- to the closed holoform by several steps with the accumulation of at least three kinetic intermediates. All these steps are accompanied by proton losses, probably occurring from the protein ligands and the side-chains involved in the interdomain H-bonding nets. The minor bihydroxamatoFe(III) species Fe(AHA)2 exchanges its iron with the C-site of apotransferrin in interaction with bicarbonate without detectable formation of any intermediate protein-iron-ligand mixed complex; direct second-order rate constant k1=4.15(+/-0.05)x10(7) M(-1) s(-1). The kinetic product loses a single proton and undergoes a modification in its conformation followed by the loss of two or three protons; first-order rate constant k2=3.25(+/-0.15) s(-1). This induces a new modification in the conformation; first-order rate constant k3=5.90(+/-0.30)x10(-2) s(-1). This new modification in conformation rate controls iron uptake by the N-site of the protein and is followed by a single proton loss; K3a=6.80 nM. Finally, the holoprotein or the monoferric transferrin in its thermodynamic equilibrated state is produced by a last modification in the conformation occurring in about 4000 seconds. But for the Fe(AHA)3 dissociation and the involvement of Fe(AHA)2 in the first step of iron uptake, this mechanism is identical to that reported for iron uptake from FeNAc3. This implies that the exchange of iron between a chelate and serum-transferrin occurs by a single general mechanism. The nature of the iron-providing chelate is only important for the first kinetic step of the exchange, which can be slowed to such an extent that it rate limits the exchange of iron.     

Gallium uptake by transferrin and interaction with receptor 1

The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate in about 50 s to yield an intermediate complex with an equilibrium constant K ₁ = (3.9 ± 1.2) × 10⁻², a direct second-order rate constant k ₁ = 425 ± 50 M⁻¹ s⁻¹ and a reverse second-order rate constant k ₋₁ = (1.1 ± 3) × 10⁴ M⁻¹ s⁻¹. The intermediate complex loses a single proton with proton dissociation constant K _1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant K _d = 1.10 ± 0.12 μM and a second-order rate constant k _d = (1.15 ± 0.3) × 10¹⁰ M⁻¹ s⁻¹. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition pathway is discussed.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

The mechanism of iron release from the transferrin-receptor 1 adduct

We report the determination in cell-free assays of the mechanism of iron release from the N-lobe and C-lobe of human serum transferrin in interaction with intact transferrin receptor 1 at 4.3< or =pH< or =6.5. Iron is first released from the N-lobe in the tens of milliseconds range and then from the C-lobe in the hundreds of seconds range. In both cases, iron loss is rate-controlled by slow proton transfers, rate constant for the N-lobe k(1)=1.20(+/-0.05)x10(6)M(-1)s(-1) and for the C-lobe k(2)=1.6(+/-0.1)x10(3)M(-1)s(-1). This iron loss is subsequent to a fast proton-driven decarbonation and is followed by two proton gains, (pK(1a))/2=5.28 per proton for the N-lobe and (pK(2a))/2=5.10 per proton for the C-lobe. Under similar experimental conditions, iron loss is about 17-fold faster from the N-lobe and is at least 200-fold faster from the C-lobe when compared to holotransferrin in the absence of receptor 1. After iron release, the apotransferrin-receptor adduct undergoes a slow partial dissociation controlled by a change in the conformation of the receptor; rate constant k(3)=1.7(+/-0.1)x10(-3)s(-1). At endosomic pH, the final equilibrated state is attained in about 1000 s, after which the free apotransferrin, two prototropic species of the acidic form of the receptor and apotransferrin interacting with the receptor coexist simultaneously. However, since recycling of the vesicle containing the receptor to the cell surface takes a few minutes, the major part of transferrin will still be forwarded to the biological fluid in the form of the apotransferrin-receptor protein-protein adduct.     

Transferrin's mechanism of interaction with receptor 1

The kinetics and thermodynamics of the interactions of transferrin receptor 1 with holotransferrin and apotransferrin in neutral and mildly acidic media are investigated at 37 degrees C in the presence of CHAPS micelles. Receptor 1 interacts with CHAPS in a very fast kinetic step (<1 micros). This is followed in neutral media by the interaction with holotransferrin which occurs in two steps after receptor deprotonation, with a proton dissociation constant (K(1a)) of 10.0 +/- 1.5 nM. The first step is detected by the T-jump technique and is associated with a molecular interaction between the receptor and holotransferrin. It occurs with a first-order rate constant (k(-1)) of (1.6 +/- 0.2) x 10(4) s(-1), a second-order rate constant (k(1)) of (3.20 +/- 0.2) x 10(10) M(-1) s(-1), and a dissociation constant (K(1)) of 0.50 +/- 0.07 microM. This step is followed by a slow change in the conformation with a relaxation time (tau(2)) of 3400 +/- 400 s and an equilibrium constant (K(2)) of (4.6 +/- 1.0) x 10(-3) with an overall affinity of the receptor for holotransferrin [(K'1)(-1)] of (4.35 +/- 0.60) x 10(8) M(-1). Apotransferrin does not interact with receptor 1 in neutral media, between pH 4.9 and 6, it interacts with the receptor in two steps after a receptor deprotonation (K(2a) = 2.30 +/- 0.3 microM). The first step occurs in the range of 1000-3000 s. It is ascribed to a slow change in the conformation which rate-controls a fast interaction between apotransferrin and receptor 1 with an overall affinity constant [(K(3))(-1)] of (2.80 +/- 0.30) x 10(7) M(-1). These results imply that receptor 1 probably exists in at least two forms, the neutral species which interacts with holotransferrin and not with apotransferrin and the acidic species which interacts with apotransferrin. At first, the interaction of the neutral receptor with holotransferrin is extremely fast. It is followed by the slow change in conformation, which leads to an important stabilization of the thermodynamic structure. In the acidic media of the endosome, the interaction of apotransferrin with the acidic receptor is sufficiently strong and rate-controlled by a very slow change in conformation which allows recycling back to the plasma membrane.     

Transferrins: iron release from lactoferrin

Iron loss in vitro by the iron scavenger bovine lactoferrin was investigated in acidic media in the presence of three different monoanions (NO(3)(-), Cl(-) and Br(-)) and one dianion (SO(4)(2-)). Holo and monoferric C-site lactoferrins lose iron in acidic media (pH< or =3.5) by a four-step mechanism. The first two steps describe modifications in the conformation affecting the whole protein, which occur also with apolactoferrin. These two processes are independent of iron load and are followed by a third step consisting of the gain of two protons. This third step is kinetically controlled by the interaction with two Cl(-), Br(-) and NO(3)(-) or one SO(4)(2-). In the fourth step, iron loss is under the kinetic control of a slow gain of two protons; third-order rate-constants k(2), 4.3(+/-0.2)x10(3), 3.4(+/-0.5)x10(3), 3.3(+/-0.5)x10(3) and 1.5(+/-0.5)x10(3) M(-2) s(-1) when the protein is in interaction with SO(4)(2-), NO(3)(-), Cl(-) or Br(-), respectively. This step is accompanied by the loss of the interaction with the anions; equilibrium constant K(2), 20+/-5 mM, 1.0(+/-0.2)x10(-1), 1.5(+/-0.5)x10(-1) and 1.0(+/-0.3)x10(-1) M(2), for SO(4)(-), NO(3)(-), Cl(-) and Br(-), respectively. This mechanism is very different from that determined in mildly acidic media at low ionic strength (micro<0.5) for the iron transport proteins, serum transferrin and ovotransferrin, with which no prior change in conformation or interaction with anions is required. These differences may result from the fact that in the transport proteins, the interdomain hydrogen bonds that consolidate the closed conformation of the iron-binding cleft occur between amino acid side-chain residues that can protonate in mildly acidic media. With bovine lactoferrin, most of the interdomain hydrogen bonds involved in the C-site and one of those involved in the N-site occur between amino acid side-chain residues that cannot protonate. The breaking of the interdomain H-bond upon protonation can trigger the opening of the iron cleft, facilitating iron loss in serum transferrin and ovotransferrin. This situation is, however, different in lactoferrin, where iron loss requires a prior change in conformation. This can explain why lactoferrin does not lose its iron load in acidic media and why it is not involved in iron transport in acidic endosomes.     

Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin

His349 in human transferrin (hTF) is a residue critical to transferrin receptor (TFR)-stimulated iron release from the C-lobe. To evaluate the importance of His349 on the TFR interaction, it was replaced by alanine, aspartate, lysine, leucine, tryptophan, and tyrosine in a monoferric C-lobe hTF construct (Fe_ChTF). Using a stopped-flow spectrofluorimeter, we determined rate processes assigned to iron release and conformational events (in the presence and in the absence of the TFR). Significantly, all mutant/TFR complexes feature dampened iron release rates. The critical contribution of His349 is most convincingly revealed by analysis of the kinetics as a function of pH (5.6–6.2). The Fe_ChTF/TFR complex titrates with a pK _a of approximately 5.9. By contrast, the H349A mutant/TFR complex releases iron at higher pH with a profile that is almost the inverse of that of the control complex. At the putative endosomal pH of 5.6 (in the presence of salt and chelator), iron is released from the H349W mutant/TFR and H349Y mutant/TFR complexes with a single rate constant similar to the iron release rate constant for the control; this suggests that these substitutions bypass the required pH-induced conformational change allowing the C-lobe to directly interact with the TFR to release iron. The H349K mutant proves that although the positive charge is crucial to complete iron release, the geometry at this position is also critical. The H349D mutant shows that a negative charge precludes complete iron release at pH 5.6 both in the presence and in the absence of the TFR. Thus, histidine uniquely drives the pH-induced conformational change in the C-lobe required for TFR interaction, which in turn promotes iron release.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Interaction kinetics of tetramethylrhodamine transferrin with human transferrin receptor studied by fluorescence correlation spectroscopy.

We applied fluorescence correlation spectroscopy (FCS) to characterize the interaction dynamics of fluorescence-labeled transferrin with transferrin receptor (hTfR) associates isolated from human placenta. The dissociation constant for the equilibrium binding of TMR-labeled ferri-transferrin to hTfR in detergent free solution was determined to be 7 +/- 3 nM. Binding curves were compatible with equal and independent binding sites present on the hTfR associates. Under pseudo-first-order conditions, with respect to transferrin, complex formation is monophasic. From these curves, association and dissociation rate constants for a reversible bimolecular binding reaction were determined, with (1.1 +/- 0.1) x 10(4) M-1 s-1 for the former and (6 +/- 4) x 10(-)4 s-1 for the latter. In dissociation exchange experiments, biphasic curves and concentration-independent reciprocal relaxation times were determined. From isothermal titration calorimetry experiments, we obtained an enthalpy change of -44.4 kJ/mol associated with the reaction. We thus conclude that the reaction is mainly enthalpy driven.     

Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing

HFE and transferrin receptor 2 (TFR2) are membrane proteins integral to mammalian iron homeostasis and associated with human hereditary hemochromatosis. Here we demonstrate that HFE and TFR2 interact in cells, that this interaction is not abrogated by disease-associated mutations of HFE and TFR2, and that TFR2 competes with TFR1 for binding to HFE. We propose a new model for the mechanism of iron status sensing that results in the regulation of iron homeostasis.     

A competitive fluorescence assay to measure the reactivity of compounds

A sensitive competitive method was developed for assessing the reactivity of compounds toward glutathione and toward thiols in general. The method employs the reaction of the fluorogenic reagent fluorescein-5-maleimide (FM) with glutathione (GSH) to generate a large increase in fluorescence emission. When the reaction is measured in the presence of a compound that competes with FM toward GSH, the rate constant for fluorescent product formation increases while the total amount of product formed at the end of the reaction decreases. These changes in the presence of a series of competitor concentrations allow one to calculate the rate constant of the reaction of the competitor with GSH. At 23 degrees C, pH 7.40 in PBS buffer the second-order rate constant of the FM-GSH reaction is k2 = (1.67 +/- 0.32) x 10(4) M(-1) x s(-1). Two GSH-reactive compounds were evaluated: the second-order rate constant for the reaction of PNU-27707 with GSH under our experimental conditions is k(i) = 5660 +/- 266 M(-1) x s(-1), while that of PNU-37802 is k(i) = 21,200 +/- 1600 M(-1) x s(-1). The method is easily adaptable to a high-throughput screening format.     

Kinetic analysis of the mass transport limited interaction between the tyrosine kinase lck SH2 domain and a phosphorylated peptide studied by a new cuvette-based surface plasmon resonance instrument

We explored the use of a newly developed cuvette-based surface plasmon resonance (SPR) instrument (IBIS) to study peptide-protein interactions. We studied the interaction between the SH2 domain of lck and a phosphotyrosine peptide EPQY*EEIPIYL which was immobilized on a sensor chip. No indications for mass transport limitation (MTL) were observed when standard kinetic approaches were used. However, addition of competing peptide during dissociation revealed a high extent of rebinding. A dissociation rate constant (k(d)) of 0.6+/-0.1 s(-1) was obtained in the presence of large amounts of peptide. A simple bimolecular binding model, applying second-order kinetics for the cuvette system, could not adequately describe the data. Fits were improved upon including a step in the model which describes diffusion of the SH2 domain from the bulk to the sensor, especially for a surface with high binding capacity. From experiments in glycerol-containing buffers, it appeared that the diffusion rate decreased with higher viscosity. It is demonstrated that MTL during association and dissociation can be described by the same diffusion rate. A binding constant (K(D)) of 5.9+/-0.8 nM was obtained from the SPR equilibrium signals by fitting to a Langmuir binding isotherm, with correction for loss of free analyte due to binding. An association rate constant k(a) of 1.1(+/-0.2)x10(8) M(-1) x s(-1) was obtained from k(d)/K(D). The values for k(a) and k(d) obtained in this way were 2-3 orders larger than that from standard kinetic analysis, ignoring MTL. We conclude that in a cuvette the extent of MTL is comparable to that in a flow system.     

The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression

Hemochromatosis is caused by mutations in HFE, a protein that competes with transferrin (TF) for binding to transferrin receptor 1 (TFR1). We developed mutant mouse strains to gain insight into the role of the Hfe/Tfr1 complex in regulating iron homeostasis. We introduced mutations into a ubiquitously expressed Tfr1 transgene or the endogenous Tfr1 locus to promote or prevent the Hfe/Tfr1 interaction. Under conditions favoring a constitutive Hfe/Tfr1 interaction, mice developed iron overload attributable to inappropriately low expression of the hormone hepcidin. In contrast, mice carrying a mutation that interferes with the Hfe/Tfr1 interaction developed iron deficiency associated with inappropriately high hepcidin expression. High-level expression of a liver-specific Hfe transgene in Hfe-/- mice was also associated with increased hepcidin production and iron deficiency. Together, these models suggest that Hfe induces hepcidin expression when it is not in complex with Tfr1.     

Indirect determination of the rate of iron uptake into the apoprotein of the ribonucleotide reductase of E. coli

The second-order rate constant k(apo) for uptake of FeII by the apoprotein of Ribonucleotide Reductase R2 has been measured by letting that reaction compete with the uptake of FeII by ferrozine (rate constant k(Fz)). The rate of the FeII/ferrozine reaction was studied at high ferrozine concentrations, and an effective first-order rate constant k(Fz) for the disappearance of FeII determined in the presence of bovine serum albumin as a viscogen. Solutions of apoprotein and ferrozine in various ratios were mixed with FeII solutions in a stopped-flow apparatus, and the growth of the 562 nm FeII(ferrozine)3 absorbance monitored. Attempts to fit the data to a variety of kinetic schemes imply that uptake of the second FeII by apo is slower than uptake of the first, suggesting that the rate-determining step in the activation of R2 is a conformational change after the uptake of the first iron. The resulting value of k(apo) is 1.8(1) x 10(6) M(-1) x s(-1).     

Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release

Efficient delivery of iron is critically dependent on the binding of diferric human serum transferrin (hTF) to its specific receptor (TFR) on the surface of actively dividing cells. Internalization of the complex into an endosome precedes iron removal. The return of hTF to the blood to continue the iron delivery cycle relies on the maintenance of the interaction between apohTF and the TFR after exposure to endosomal pH (≤6.0). Identification of the specific residues accounting for the pH-sensitive nanomolar affinity with which hTF binds to TFR throughout the cycle is important to fully understand the iron delivery process. Alanine substitution of 11 charged hTF residues identified by available structures and modeling studies allowed evaluation of the role of each in (1) binding of hTF to the TFR and (2) TFR-mediated iron release. Six hTF mutants (R50A, R352A, D356A, E357A, E367A, and K511A) competed poorly with biotinylated diferric hTF for binding to TFR. In particular, we show that Asp356 in the C-lobe of hTF is essential to the formation of a stable hTF-TFR complex: mutation of Asp356 in the monoferric C-lobe hTF background prevented the formation of the stoichiometric 2:2 (hTF:TFR monomer) complex. Moreover, mutation of three residues (Asp356, Glu367, and Lys511), whether in the diferric or monoferric C-lobe hTF, significantly affected iron release when in complex with the TFR. Thus, mutagenesis of charged hTF residues has allowed identification of a number of residues that are critical to formation of and release of iron from the hTF-TFR complex.     

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II

The bis(terpyridine)cobalt(II), Co(terpy)2(2+), reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 degrees C. Co(terpy)2(2+) initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0+/-0.4x10(6) M(-1)s(-1) at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s(-1) was observed for the reduction of CcP-I by 79 microM Co(terpy)2(2+) at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)2(2+) shows saturation kinetics with a maximum observed rate constant, k3max, of 24+/-2 s(-1) at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)2(2+) reduction of CcP-II, k3, is 1.2+/-0.5x10(6) M(-1) s-1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0+/-0.8x10(5) M(-1) s(-1), 1.2+/-0.5x10(5) M(-1) s(-1), and 11+/-3 s(-1), respectively. Both the product, Co(terpy)2(3+), and ferricytochrome c inhibit the rate of Co(terpy)2(2+) reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)2(3+) molecules bind to the native enzyme in low ionic strength buffers.     

Spectral and kinetic studies of the oxidation of monosubstituted phenols and anilines by recombinant Synechocystis catalase-peroxidase compound I.

A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803 is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by a bifunctional catalase-peroxidase compound I using the sequential-mixing stopped-flow technique. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. A >/=50-fold excess of peroxoacetic acid is required to obtain a spectrum of relatively pure and stable compound I which is characterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbestic points between the native enzyme and compound I at 357 and 430 nm. The apparent second-order rate constant for formation of compound I from ferric enzyme and peroxoacetic acid is (8.74 +/- 0.26) x 10(3) M(-)(1) s(-)(1) at pH 7. 0. Reduction of compound I by aromatic donor molecules is dependent upon the substituent effect on the benzene ring. The apparent second-order rate constants varied from (3.6 +/- 0.1) x 10(6) M(-)(1) s(-)(1) for p-hydroxyaniline to (5.0 +/- 0.1) x 10(2) M(-)(1) s(-)(1) for p-hydroxybenzenesulfonic acid. They are shown to correlate with the substituent constants in the Hammett equation, which suggests that in bifunctional catalase-peroxidases the aromatic donor molecule donates an electron to compound I and loses a proton simultaneously. The value of rho, the susceptibility factor in the Hammett equation, is -3.4 +/- 0.4 for the phenols and -5.1 +/- 0.8 for the anilines. The pH dependence of compound I reduction by aniline exhibits a relatively sharp maximum at pH 5. The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.