-
[show abstract]
[hide abstract]
ABSTRACT: The recent crystal structure of two monoferric human serum transferrin (Fe(N)hTF) molecules bound to the soluble portion of the homodimeric transferrin receptor (sTFR) has provided new details about this binding interaction that dictates the delivery of iron to cells. Specifically, substantial rearrangements in the homodimer interface of the sTFR occur as a result of the binding of the two Fe(N)hTF molecules. Mutagenesis of selected residues in the sTFR highlighted in the structure was undertaken to evaluate the effect on function. Elimination of Ca(2+) binding in the sTFR by mutating two of four coordinating residues ([E465A,E468A]) results in low production of an unstable and aggregated sTFR. Mutagenesis of two histidines ([H475A,H684A]) at the dimer interface had little effect on the kinetics of release of iron at pH 5.6 from either lobe, reflecting the inaccessibility of this cluster to solvent. Creation of an H318A sTFR mutant allows assignment of a small pH-dependent initial decrease in the magnitude of the fluorescence signal to His318. Removal of the four C-terminal residues of the sTFR, Asp757-Asn758-Glu759-Phe760, eliminates pH-stimulated release of iron from the C-lobe of the Fe(2)hTF/sTFR Δ757-760 complex. The inability of this sTFR mutant to bind and stabilize protonated hTF His349 (a pH-inducible switch) in the C-lobe of hTF accounts for the loss. Collectively, these studies support a model in which a series of pH-induced events involving both TFR residue His318 and hTF residue His349 occurs to promote receptor-stimulated release of iron from the C-lobe of hTF.
Biochemistry 03/2012; 51(10):2113-21. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemistry 12/2011; 51(2):686-94. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Delivery of iron to cells requires binding of two iron-containing human transferrin (hTF) molecules to the specific homodimeric transferrin receptor (TFR) on the cell surface. Through receptor-mediated endocytosis involving lower pH, salt, and an unidentified chelator, iron is rapidly released from hTF within the endosome. The crystal structure of a monoferric N-lobe hTF/TFR complex (3.22-Å resolution) features two binding motifs in the N lobe and one in the C lobe of hTF. Binding of Fe(N)hTF induces global and site-specific conformational changes within the TFR ectodomain. Specifically, movements at the TFR dimer interface appear to prime the TFR to undergo pH-induced movements that alter the hTF/TFR interaction. Iron release from each lobe then occurs by distinctly different mechanisms: Binding of His349 to the TFR (strengthened by protonation at low pH) controls iron release from the C lobe, whereas displacement of one N-lobe binding motif, in concert with the action of the dilysine trigger, elicits iron release from the N lobe. One binding motif in each lobe remains attached to the same α-helix in the TFR throughout the endocytic cycle. Collectively, the structure elucidates how the TFR accelerates iron release from the C lobe, slows it from the N lobe, and stabilizes binding of apohTF for return to the cell surface. Importantly, this structure provides new targets for mutagenesis studies to further understand and define this system.
Proceedings of the National Academy of Sciences 08/2011; 108(32):13089-94. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe(3+) and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated.
We provide an overview of the kinetics of iron release from hTF±the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided.
Delivery of Fe(3+)to actively dividing cells by hTF is essential; too much or too little Fe(3+) directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential.
Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe(3+) release within a physiologically relevant time frame. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders.
Biochimica et Biophysica Acta 06/2011; 1820(3):326-33. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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(C)hTF). 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(C)hTF/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.
European Journal of Biochemistry 11/2010; 15(8):1341-52. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe.
Protein Expression and Purification 07/2010; 72(1):32-41. · 1.59 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human serum transferrin (hTF) binds two ferric iron ions which are delivered to cells in a transferrin receptor (TFR) mediated process. Critical to the delivery of iron to cells is the binding of hTF to the TFR and the efficient release of iron orchestrated by the interaction. Within the endosome, iron release from hTF is also aided by lower pH, the presence of anions, and a chelator yet to be identified. We have recently shown that three of the four residues comprising a loop in the N-lobe (Pro142, Lys144, and Pro145) are critical to the high-affinity interaction of hTF with the TFR. In contrast, Arg143 in this loop does not participate in the binding isotherm. In the current study, the kinetics of iron release from alanine mutants of each of these four residues (placed into both diferric and monoferric N-lobe backgrounds) have been determined +/- the TFR. The R143A mutation greatly retards the rate of iron release from the N-lobe in the absence of the TFR but has considerably less of an effect in its presence. Our data definitively show that Arg143 serves as a kinetically significant anion binding (KISAB) site that is, by definition, sensitive to salt concentration and critical to the conformational change necessary to induce iron release from the N-lobe of hTF (in the absence of the TFR). This is the first identification of an authentic KISAB site in the N-lobe of hTF. The effect of the single R143A mutation on the kinetic profile of iron release provides a dramatic illustration of the dynamic nature of hTF.
Biochemistry 05/2010; 49(19):4200-7. · 3.42 Impact Factor
-
N Dennis Chasteen
Biochimica et Biophysica Acta 05/2010; 1800(8):689-90. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.
Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.
More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe(2+) to apoHuHF produces iron core particle size diameters from 3.8 + or - 0.3 to 6.2 + or - 0.3 nm. Diameters from 3.4 + or - 0.6 to 6.5 + or - 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts approximately 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M=484,120 g/mol, nu=0.735 mL/g, s(20,w)=17.0 S and D(20,w)=3.21 x 10(-)(7) cm(2)/s; and for apoHuHF M=506,266 g/mol, nu=0.724 mL/g, s(20,w)=18.3S and D(20,w)=3.18 x 10(-)(7) cm(2)/s.
The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals.
Biochimica et Biophysica Acta 03/2010; 1800(8):858-70. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transferrins are a family of bilobal iron-binding proteins that play the crucial role of binding ferric iron and keeping it in solution, thereby controlling the levels of this important metal. Human serum transferrin (hTF) carries one iron in each of two similar lobes. Understanding the detailed mechanism of iron release from each lobe of hTF during receptor-mediated endocytosis has been extremely challenging because of the active participation of the transferrin receptor (TFR), salt, a chelator, lobe-lobe interactions, and the low pH within the endosome. Our use of authentic monoferric hTF (unable to bind iron in one lobe) or diferric hTF (with iron locked in one lobe) provided distinct kinetic end points, allowing us to bypass many of the previous difficulties. The capture and unambiguous assignment of all kinetic events associated with iron release by stopped-flow spectrofluorimetry, in the presence and in the absence of the TFR, unequivocally establish the decisive role of the TFR in promoting efficient and balanced iron release from both lobes of hTF during one endocytic cycle. For the first time, the four microscopic rate constants required to accurately describe the kinetics of iron removal are reported for hTF with and without the TFR. Specifically, at pH 5.6, the TFR enhances the rate of iron release from the C-lobe (7-fold to 11-fold) and slows the rate of iron release from the N-lobe (6-fold to 15-fold), making them more equivalent and producing an increase in the net rate of iron removal from Fe(2)hTF. Calculated cooperativity factors, in addition to plots of time-dependent species distributions in the absence and in the presence of the TFR, clearly illustrate the differences. Accurate rate constants for the pH and salt-induced conformational changes in each lobe precisely delineate how delivery of iron within the physiologically relevant time frame of 2 min might be accomplished.
Journal of Molecular Biology 11/2009; 396(1):130-40. · 4.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Transferrin (TF) is a bilobal transport protein that acquires ferric iron from the diet and holds it tightly within the cleft of each lobe (thereby preventing its hydrolysis). The iron is delivered to actively dividing cells by receptor mediated endocytosis in which diferric TF preferentially binds to TF receptors (TFRs) on the cell surface and the entire complex is taken into an acidic endosome. A combination of lower pH, a chelator, inorganic anions, and the TFR leads to the efficient release of iron from each lobe. Identification of residues/regions within both TF and TFR required for high affinity binding has been an ongoing goal in the field. In the current study, we created human TF (hTF) mutants to identify a region critical to the interaction with the TFR which also constitutes part of an overlapping epitope for two monoclonal antibodies (mAbs) to the N-lobe, one of which was previously shown to block binding of hTF to the TFR. Four single point mutants, P142A, R143A, K144A, and P145A in the N-lobe, were placed into diferric hTF. Isothermal titration calorimetry (ITC) revealed that three of the four residues (Pro142, Lys144, and Pro145) in this loop are essential to TFR binding. Additionally, Lys144 is common to the recognition of both mAbs which show different sensitivities to the three other residues. Taken together these studies prove that this loop is required for binding of the N-lobe of hTF to the TFR, provide a more precise description of the role of each residue in the loop in the interaction with the TFR, and confirm that the N-lobe is essential to high affinity binding of diferric hTF to TFR.
Journal of Molecular Recognition 08/2009; 22(6):521-9. · 3.31 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Iron stored in phytoferritin plays an important role in the germination and early growth of seedlings. The protein is located
in the amyloplast where it stores large amounts of iron as a hydrated ferric oxide mineral core within its shell-like structure.
The present work was undertaken to study alternate mechanisms of core formation in pea seed ferritin (PSF). The data reveal
a new mechanism for mineral core formation in PSF involving the binding and oxidation of iron at the extension peptide (EP)
located on the outer surface of the protein shell. This binding induces aggregation of the protein into large assemblies of
∼400 monomers. The bound iron is gradually translocated to the mineral core during which time the protein dissociates back
into its monomeric state. Either the oxidative addition of Fe2+ to the apoprotein to form Fe3+ or the direct addition of Fe3+ to apoPSF causes protein aggregation once the binding capacity of the 24 ferroxidase centers (48 Fe3+/shell) is exceeded. When the EP is enzymatically deleted from PSF, aggregation is not observed, and the rate of iron oxidation
is significantly reduced, demonstrating that the EP is a critical structural component for iron binding, oxidation, and protein
aggregation. These data point to a functional role for the extension peptide as an iron binding and ferroxidase center that
contributes to mineralization of the iron core. As the iron core grows larger, the new pathway becomes less important, and
Fe2+ oxidation and deposition occurs directly on the surface of the iron core.
Journal of Biological Chemistry 06/2009; 284(25):16743-16751. · 4.77 Impact Factor
-
Anne B Mason,
Peter J Halbrooks,
Nicholas G James,
Shaina L Byrne,
John K Grady, N Dennis Chasteen,
Cedric E Bobst,
Igor A Kaltashov,
Valerie C Smith,
Ross T A MacGillivray,
Stephen J Everse
[show abstract]
[hide abstract]
ABSTRACT: The G65R mutation in the N-lobe of human transferrin was created to mimic a naturally occurring variant (G394R) found in the homologous C-lobe. Because Gly65 is hydrogen-bonded to the iron-binding ligand Asp63, it comprises part of the second-shell hydrogen bond network surrounding the iron within the metal-binding cleft of the protein. Substitution with an arginine residue at this position disrupts the network, resulting in much more facile removal of iron from the G65R mutant. As shown by UV-vis and EPR spectroscopy, and by kinetic assays measuring the release of iron, the G65R mutant can exist in three forms. Two of the forms (yellow and pink in color) are interconvertible. The yellow form predominates in 1 M bicarbonate; the pink form is generated from the yellow form upon exchange into 1 M HEPES buffer (pH 7.4). The third form (also pink in color) is produced by the addition of Fe(3+)-(nitrilotriacetate)(2) to apo-G65R. Hydrogen-deuterium exchange experiments are consistent with all forms of the G65R mutant assuming a more open conformation. Additionally, mass spectrometric analysis reveals the presence of nitrilotriacetate in the third form. The inability to obtain crystals of the G65R mutant led to development of a novel crystallization strategy in which the G65R/K206E double mutation stabilizes a single closed pink conformer and captures Arg65 in a single position. Collectively, these studies highlight the importance of the hydrogen bond network in the cleft, as well as the inherent flexibility of the N-lobe which, although able to adapt to accommodate the large arginine substitution, exists in multiple conformations.
Biochemistry 03/2009; 48(9):1945-53. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ferritin is a widespread iron mineralizing and detoxification protein that stores iron as a hydrous ferric oxide mineral core within a shell-like structure of 4/3/2 octahedral symmetry. Iron mineralization is initiated at dinuclear ferroxidase centers inside the protein where Fe(2+) and O(2) meet and react to form a mu-1,2-peroxodiferric intermediate that subsequently decays to form mu-oxo dimeric and oligomeric iron(III) species and ultimately the mineral core. Several types of channels penetrate the protein shell and are possible pathways for the diffusion of iron and dioxygen to the ferroxidase centers. In the present study, UV/visible and fluorescence stopped-flow spectrophotometries were used to determine the kinetics and pathways of Fe(2+) diffusion into the protein shell, its binding at the ferroxidase center and its subsequent oxidation by O(2). Three fluorescence variants of human H-chain ferritin were prepared in which Trp34 was introduced near the ferroxidase center. They included a control variant no. 1 (W93F/Y34W), a "1-fold" channel variant no. 2 (W93F/Y34W/Y29Q) and a 3-fold channel variant no. 3 (Y34W/W93F/D131I/E134F). Anaerobic rapid mixing of Fe(2+) with apo-variant no. 1 quenched the fluorescence of Trp34 with a rate exhibiting saturation kinetics with respect to Fe(2+) concentration, consistent with a process involving facilitated diffusion. A half-life of approximately 3 ms for this process is attributed to the time for diffusion of Fe(2+) across the protein shell to the ferroxidase center. No fluorescence quenching was observed with the 3-fold channel variant no. 3 or when Zn(2+) was prebound in each of the eight 3-fold channels of variant no. 1, observations indicating that the hydrophilic channels are the only avenues for rapid Fe(2+) access to the ferroxidase center. Substitution of Tyr29 with glutamine at the entrance of the "1-fold" hydrophobic channel had no effect on the rate of Fe(2+) oxidation to form the mu-1,2-peroxodiferric complex (t(1/2) approximately 38 ms), a finding demonstrating that Tyr29 and, by inference, the "1-fold" channels do not facilitate O(2) transport to the ferroxidase center, contrary to predictions of DFT and molecular dynamics calculations. O(2) diffusion into ferritin occurs on a time scale that is fast relative to the millisecond kinetics of the stopped-flow experiment.
Journal of the American Chemical Society 01/2009; 130(52):17801-11. · 9.91 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Electron paramagnetic resonance (EPR) signals at g' = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe3+ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g' = 4.3 intensity standard and for accurately quantifying the amount of Fe3+ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308-315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe3+-EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu2+-EDTA and aqua VO2+, both S = 1/2 ions, and of Fe3+-transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of +/-3.6, +/-5.3 and +/-2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe3+-desferrioxamine of known Fe3+ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.
JBIC Journal of Biological Inorganic Chemistry 02/2008; 13(1):15-24. · 3.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ferritins are ubiquitous iron storage and detoxification proteins distributed throughout the plant and animal kingdoms. Mammalian ferritins oxidize and accumulate iron as a ferrihydrite mineral within a shell-like protein cavity. Iron deposition utilizes both O(2) and H(2)O(2) as oxidants for Fe(2+) where oxidation can occur either at protein ferroxidase centers or directly on the surface of the growing mineral core. The present study was undertaken to determine whether the nature of the mineral core formed depends on the protein ferroxidase center versus mineral surface mechanism and on H(2)O(2) versus O(2) as the oxidant. The data reveal that similar cores are produced in all instances, suggesting that the structure of the core is thermodynamically, not kinetically controlled. Cores averaging 500 Fe/protein shell and diameter approximately 2.6 nm were prepared and exhibited superparamagnetic blocking temperatures of 19 and 22 K for the H(2)O(2) and O(2) oxidized samples, respectively. The observed blocking temperatures are consistent with the unexpectedly large effective anisotropy constant K(eff)=312 kJ/m(3) recently reported for ferrihydrite nanoparticles formed in reverse micelles [E.L. Duarte, R. Itri, E. Lima Jr., M.S. Batista, T.S. Berquó and G.F. Goya, Large Magnetic Anisotropy in ferrihydrite nanoparticles synthesized from reverse micelles, Nanotechnology 17 (2006) 5549-5555.]. All ferritin samples exhibited two magnetic phases present in nearly equal amounts and ascribed to iron spins at the surface and in the interior of the nanoparticle. At 4.2 K, the surface spins exhibit hyperfine fields, H(hf), of 436 and 445 kOe for the H(2)O(2) and O(2) samples, respectively. As expected, the spins in the interior of the core exhibit larger H(hf) values, i.e. 478 and 486 kOe for the H(2)O(2) and O(2) samples, respectively. The slightly smaller hyperfine field distribution DH(hf) for both surface (78 kOe vs. 92 kOe) and interior spins (45 kOe vs. 54 kOe) of the O(2) sample compared to the H(2)O(2) samples implies that the former is somewhat more crystalline.
Biophysical Chemistry 12/2007; 130(3):114-21. · 2.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Green fluorescent proteins (GFP) are widely used in vivo molecular markers. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O2*-)) and exhibits SOD-like activity by competing with cytochrome c for reaction with O2*-. When exposed to high amounts of O2*- the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins, can provide supplementary antioxidant protection.
Biochimica et Biophysica Acta 12/2006; 1760(11):1690-5. · 4.66 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Good's zwitterionic buffers are widely used in biological and biochemical research in which hydrogen peroxide is a solution component. This study was undertaken to determine whether Good's buffers exhibit reactivity toward H(2)O(2). It is found that H(2)O(2) oxidizes both morpholine ring-containing buffers (e.g., Mops, Mes) and piperazine ring-containing zwitterionic buffers (e.g., Pipes, Hepes, and Epps) to produce their corresponding N-oxide forms. The percentage of oxidized buffer increases as the concentration of H(2)O(2) increases. However, the rate of oxidation is relatively slow. For example, no oxidized Mops was detected 2h after adding 0.1M H(2)O(2) to 0.1M Mops (pH 7.0), and only 5.7% was oxidized after 24h exposure to H(2)O(2). Thus, although all of these buffers can be oxidized by H(2)O(2), their slow reaction does not significantly perturb levels of H(2)O(2) in the time frame and at the concentrations of most biochemical studies. Therefore, the previously reported rapid loss of H(2)O(2) produced from the ferroxidase reaction of ferritin is unlikely due to reaction of H(2)O(2) with buffer, a conclusion supported by the fact that H(2)O(2) is also lost rapidly when the solution pH of the ferroxidase reaction is controlled by a pH stat apparatus in the absence of buffer.
Analytical Biochemistry 03/2006; 349(2):262-7. · 3.00 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Overexpression of human H-chain ferritin (HuHF) is known to impart a degree of protection to cells against oxidative stress and the associated damage to DNA and other cellular components. However, whether this protective activity resides in the protein's ability to inhibit Fenton chemistry as found for Dps proteins has never been established. Such inhibition does not occur with the related mitochondrial ferritin which displays much of the same iron chemistry as HuHF, including an Fe(II)/H(2)O(2) oxidation stoichiometry of approximately 2:1. In the present study, the ability of HuHF to attenuate hydroxyl radical production by the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + OH(-) + *OH) was examined by electron paramagnetic resonance (EPR) spin-trapping methods. The data demonstrate that the presence of wild-type HuHF during Fe(2+) oxidation by H(2)O(2) greatly decreases the amount of .OH radical produced from Fenton chemistry whereas the ferroxidase site mutant 222 (H62K + H65G) and human L-chain ferritin (HuLF) lack this activity. HuHF catalyzes the pairwise oxidation of Fe(2+) by the detoxification reaction [2Fe(2+) + H(2)O(2) + 2H(2)O --> 2Fe(O)OH(core) + 4H(+)] that occurs at the ferroxidase site of the protein, thereby preventing the production of hydroxyl radical. The small amount of *OH radical that is produced in the presence of ferritin (<or=1% of the iron oxidized) appears to arise from the reaction of H(2)O(2) with Fe(III) in the protein rather than from simple Fenton chemistry. The results are discussed in terms of recent experiments reporting both protective and degradative effects of ferritin iron on the integrity of nuclear DNA.
Biochemistry 03/2006; 45(10):3429-36. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.
Biochemistry 11/2005; 44(42):13837-46. · 3.42 Impact Factor
