Iron-bleomycin interactions with oxygen and oxygen analogues. Effects on spectra and drug activity.

Despite extensive structural dissimilarities, iron . bleomycin complexes and heme-containing oxygenases display remarkable similarities in binding oxygen antagonists and in spectral properties deriving from bound iron. Fe(II)-bleomycin reversibly forms a complex with either CO or isocyanide (lambda max = 384 and 497 nm, respectively), either of which interfere with its oxygen-dependent cleavage of DNA. A similar but paramagnetic complex forms with NO (lambda max = 470 nm; AN = 24 G). In contrast, cyanide enhances bleomycin activity against DNA. Complexes of bleomycin and FE(III), formed either by direct association or by autoxidation of the Fe(II) . bleomycin complex, exhibit indistinguishable EPR and visible spectra, which change characteristically with pH. At neutral pH, Fe(III) . bleomycin is a low spin complex (g = 2.45, 2.18, 1.89; lambda max = 365, 384 nm) and, at low pH, it is a high spin rhombic complex (geff = 9.4, 4.3; lambda max = 430 nm). These complexes are interconvertible (pK 4.3). Fe(II) . bleomycin oxidation, although reversible by spectral criteria, is accompanied by drug inactivation unless DNA is present.     

Synergistic anion-directed coordination of ferric and cupric ions to bovine serum transferrin--an inorganic perspective

A series of new iron(III) and copper(II) complexes of bovine serum transferrin (BTf), with carbonate and/or oxalate as the synergistic anion, are presented. The complexes [Fe(2)(CO(3))(2)BTf], [Fe(2)(C(2)O(4))(2)BTf], [Cu(2)(CO(3))(2)BTf] and [Cu(C(2)O(4))BTf] were prepared by standard titrimetric techniques. The oxalate derivatives were also obtained from the corresponding carbonate complexes by anion-displacement. The site-preference of the transition metal-oxalate synergism has facilitated the preparation and isolation of the mononuclear complex [Cu(C(2)O(4))BTf], the mixed-anion complexes [Cu(2)(CO(3))(C(2)O(4))BTf] and [Fe(2)(CO(3))(C(2)O(4))BTf] and the mixed-metal complex [FeCu(C(2)O(4))(2)BTf]. The sensitivity of electron paramagnetic resonance (EPR) spectroscopy to the nature of the synergistic anions at the specific-binding sites of the transferrins has made this physical technique particularly indispensable to this study. None of the other members of the transferrin family of proteins has ever been demonstrated to bind the ferric and cupric ions one after the other, each occupying a separate specific-binding site of the same transferrin molecule, as a response to the coordination restrictions imposed by the oxalate ion. The bathochromic shift of the visible p(pi)-d(pi*) CT band for iron(III)-BTf and the hypsochromic shift of the p(pi)-d(sigma*) CT band for copper(II)-BTf, on replacing carbonate by oxalate as the associated anion, are consistent with the relative positions of these anionic ligands in the spectrochemical series and the nature of the d-type acceptor orbitals involved in the CT transitions. The binding and spectroscopic properties of bovine serum transferrin--a serum transferrin--very nearly mirror those of human serum transferrin, but differ significantly from those of human lactoferrin.     

Anion binding by human lactoferrin: results from crystallographic and physicochemical studies.

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.     

Kinetics and mechanism of iron(III) complexation by ferric binding protein: the role of phosphate

Iron transport across the periplasmic space to the cytoplasmic membrane of certain Gram-negative bacteria is mediated by a ferric binding protein (Fbp). This requires Fe(3+) loading of Fbp at the inner leaflet of the outer membrane. A synergistic anion is required for tight Fe(3+) sequestration by Fbp. Although phosphate fills this role in the protein isolated from bacterial cell lysates, nitrilotriacetate anion (NTA) can also satisfy this requirement in vitro. Here, we report the kinetics and mechanism of Fe(3+) loading of Fbp from Fe(NTA)(aq) in the presence of phosphate at pH 6.5. The reaction proceeds in four kinetically distinguishable steps to produce Fe(3+)Fbp(PO(4)) as a final product. The first three steps exhibit half-lives ranging from ca. 20 ms to 0.5 min, depending on the concentrations, and produce Fe(3+)Fbp(NTA) as an intermediate product of significant stability. The rate for the first step is accelerated with an increasing phosphate concentration, while that of the third step is retarded by phosphate. Conversion of Fe(3+)Fbp(NTA) to Fe(3+)Fbp(PO(4)) in the fourth step is a slow process (half-life approximately 2 h) and is facilitated by free phosphate. A mechanism for the Fe(3+)-loading process is proposed in which the synergistic anions, phosphate and NTA, play key roles. These data suggest that not only is a synergistic anion required for tight Fe(3+) sequestration by Fbp, but also the synergistic anion plays a critical role in the process of inserting Fe(3+) into the Fbp binding site.     

The nonspecific binding of Fe3+ to transferrin in the absence of synergistic anions.

An obligatory role for barbonate (or other synergistic anions) in the specific binding of Fe3+ by transferrin has been a point of controversy for two decades. There are an equal number of confirmatory and negative reports of specific Fe3+-transferrin binary complexes. A criticism of previous studies is the use of only one synthetic route, and limited product testing. This study reports the development of several preparative routes aimed at the formation of a specific Fe3+-transferrin complex, and the characterization of the products by spectrophotometry and chemical reactivity. The preparative routes described include: (a) displacement of carbonate from Fe3+-transferrin-CO32- at low pH followed by removal of CO2 by several techniques; (b) addition of FeCl3 to apotransferrin under CO2-free conditions; (c) oxidation of Fe2+ in the presence of apotransferrin under CO2-free conditions; (d) reaction of apotransferrin with nonsubstituting Fe3+ complexes in the absence of CO2; and (e) attempts to displace anions from weak Fe3+-transferrin-anion complexes. The product were examined with regard to their visible spectra, and their examined with regard to their visible spectra, and their reactivity with: (a) NaHCO3, (b) Fe3+-nitrilotriacetic acid in NaHCO3, and (c) citrate. The results are compared with the characteristics of Fe3+-transferrin-anion complexes and nonspecific Fe3+, transferrin mixtures. The data indicate that in the absence of synergistic anions the affinity of the specific metal binding sites of transfe-rin for Fe3+ is so low as to not compete favorably with hydrolytic polymerization and nonspecific binding effects.     

O2- and alpha-ketoglutarate-dependent tyrosyl radical formation in TauD,an alpha-keto acid-dependent non-heme iron dioxygenase

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.5 min(-)(1), that transforms to yield a greenish brown chromophore (lambda(max) = 550 nm, 700 M(-)(1) x cm(-)(1)). The latter feature exhibits resonance Raman vibrations consistent with an Fe(III) catecholate species presumed to arise from enzymatic self-hydroxylation of a tyrosine residue. Significantly, (18)O labeling studies reveal that the added oxygen atom derives from solvent rather than from O(2). The transient yellow species, identified as a tyrosyl radical on the basis of EPR studies, is formed after alphaKG decomposition. Substitution of two active site tyrosine residues (Tyr73 and Tyr256) by site-directed mutagenesis identified Tyr73 as the likely site of formation of both the tyrosyl radical and the catechol-associated chromophore. The involvement of the tyrosyl radical in catalysis is excluded on the basis of the observed activity of the enzyme variants. We suggest that the Fe(IV) oxo species generally proposed (but not yet observed) as an intermediate for this family of enzymes reacts with Tyr73 when substrate is absent to generate Fe(III) hydroxide (capable of exchanging with solvent) and the tyrosyl radical, with the latter species participating in a multistep TauD self-hydroxylation reaction.     

Phosphate ester hydrolysis is catalyzed by a bacterial transferrin: potential implications for in vivo iron transport mechanisms

Two synergistic anions, p-nitrophenyl phosphate ester (NPP) and SO(4)(2-), were found to form new stable assemblies with Fe(3+) and a bacterial transferrin, FbpA (FbpA=ferric binding protein). Fe(3+)FbpA-SO(4) undergoes rapid anion exchange in the presence of NPP to form Fe(3+)FbpA-NPP. Formation of Fe(3+)FbpA-NPP was found to accelerate the rate of hydrolysis of the bound phosphate ester (k(hyd)=1.6 x 10(-6) s(-1) at 25 degrees C and pH 6.5) by >10(3) fold over the uncatalyzed reaction. These findings suggest a dual function for FbpA in vivo: transport of Fe(3+) across the periplasmic space to the inner membrane in certain gram-negative bacteria and hydrolysis of periplasmic polyphosphates.     

Ferriprotoporphyrin IX mediated oxygen activation/insertion reactions: the anerobic reduction of the aniline-Fe3+-microperoxidase-8 complex by NADH

A spectrophotometric study of the reduction of the Fe3+ microperoxidase-8-aniline (Fe3+-MP-8-An) complex has been carried out. Addition of NADH to a solution of Fe3+-MP-8-An under strictly anerobic conditions results in the formation of a species with lambda max = 414 nm (Fe3+-MP-8-An lambda max 407 nm). The kinetics of formation of this species show an induction period (tau) which follows saturation kinetics with respect to [aniline] with Km(app) = 2.2 x 10(-3) mol dm-3, i.e., close to that obtained in the preceding paper from O2 consumption kinetics mediated by MP-8. Addition of an anerobic solution of the NADH reduced MP-8-An complex, to a saturated O2 solution at pH 12 in the presence of 0.5 mM NADH and aniline 10 mM results in the virtual elimination of the induction phase, which has previously characterized O2 consumption kinetics in ferriprotoporphyrin IX oxygen activation systems. The Arrhenius activation energy for the reduction of the Fe3+-MP-8-An complex is close to that observed for the first reductive step in the cyt P-450 O2 activation cycle. Anerobic reduction of Fe3+-MP-8 by sodium dithionite in 20% MeOH/Aq at pH 8 followed by anerobic titration of the Fe2+-MP-8 (lambda max 420.5 nm) with aniline at pH 12 gives rise to a species lambda max 415 with KD for the process = 4.4 x 10(-3) mol dm-3 (+/- 1.2 x 10(-3) mol dm-3).     

Mechanistic studies of two dioxygenases in the methionine salvage pathway of Klebsiella pneumoniae

Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda(max) red shift upon binding. The dianion formation from II causes the same lambda(max) red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O(2) to form a peroxide anion intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate.     

The carbonate radical is a site-selective oxidizing agent of guanine in double-stranded oligonucleotides

The carbonate radical anion (CO(3)) is believed to be an important intermediate oxidant derived from the oxidation of bicarbonate anions and nitrosoperoxocarboxylate anions (formed in the reaction of CO(2) with ONOO(-)) in cellular environments. Employing nanosecond laser flash photolysis methods, we show that the CO(3) anion can selectively oxidize guanines in the self-complementary oligonucleotide duplex d(AACGCGAATTCGCGTT) dissolved in air-equilibrated aqueous buffer solution (pH 7.5). In these time-resolved transient absorbance experiments, the CO(3) radicals are generated by one-electron oxidation of the bicarbonate anions (HCO(3)(-)) with sulfate radical anions (SO(4)) that, in turn, are derived from the photodissociation of persulfate anions (S(2)O(8)(2-)) initiated by 308-nm XeCl excimer laser pulse excitation. The kinetics of the CO(3) anion and neutral guanine radicals, G(-H)( small middle dot), arising from the rapid deprotonation of the guanine radical cation, are monitored via their transient absorption spectra (characteristic maxima at 600 and 315 nm, respectively) on time scales of microseconds to seconds. The bimolecular rate constant of oxidation of guanine in this oligonucleotide duplex by CO(3) is (1.9 +/- 0.2) x 10(7) m(-1) s(-1). The decay of the CO(3) anions and the formation of G(-H)( small middle dot) radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.     

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

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

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

EDTA bis-(methyl tyrosinate): a chelating peptoid peroxynitrite scavenger

Conjugation of ethylenediaminetetra-acetic acid (EDTA) to methyl tyrosinate generates a chelating peptoid EDTA bis-(methyl tyrosinate), (EBMT). Peroxynitrite-mediated nitration was studied for the free peptoid and its ferric and cupric complexes. The nitration products were monitored by electronic absorption spectroscopy at lambda(max) of 420 nm (mono-nitrated) and 440 nm (di-nitrated). Peak deconvolution was effected by pH manipulation as the mono-nitrated analogue of tyrosine exhibited a bathochromic shift from 365 nm (below its pK(a) of 6.8) to 420 nm. Rates of nitration were: free peptoid 相似文献   

Kinetics of iron release from ferric binding protein (FbpA): mechanistic implications in bacterial periplasm-to-cytosol Fe3+ transport

The ferric binding protein (FbpA) transports iron across the periplasmic space of certain Gram-negative bacteria and is an important component involved in iron acquisition by pathogenic Neisseria spp. (Neisseria gonorrheae and Neisseria meningitidis). Previous work has demonstrated that the synergistic anion, required for tight Fe(3+) sequestration by FbpA, also plays a key role in inserting Fe(3+) into the FbpA binding site. Here, we investigate the iron release process from various forms of holo-FbpA, Fe(3+)FbpA-X, during the course of a chelator competition reaction using EDTA and Tiron. Fe(3+)FbpA-X represents the protein assembly complex with different synergistic anions, X = PO(4)(3)(-) and NTA. Stepwise mechanisms of Fe(3+) release are proposed on the basis of kinetic profiles of these chelator competition reactions. Fe(3+)FbpA-PO(4) and Fe(3+)FbpA-NTA react differently with EDTA and Tiron during the Fe(3+)-exchange process. EDTA replaces PO(4)(3)(-) and NTA from the first coordination shell of Fe(3+) and acts as a synergistic anion to give a spectroscopically distinguishable intermediate, Fe(3+)FbpA-EDTA, prior to pulling Fe(3+) out of the protein. Tiron, on the other hand, does not act as a synergistic anion but is a more efficient competing chelator as it removes Fe(3+) from FbpA at rate much faster than EDTA. These results reaffirm the contribution of the synergistic anion to the FbpA iron transport process as the anion, in addition to playing a facilitative role in iron binding, appears to have a "gatekeeper" role, thereby modulating the Fe(3+) release process.     

Effect of carbonate anion on cytochrome P450 2D6-mediated metabolism in vitro: the potential role of multiple oxygenating species

Studies were designed to investigate various anions and their effects on cytochrome P450 2D6-mediated metabolism in vitro. Incubations were initially performed in buffered phosphate, carbonate, sulfate, and acetate solutions (50mM, pH 7.4), with CYP2D6 substrates dextromethorphan, 7-methoxy-4-(aminomethyl)-coumarin (MAMC), (S,S)-3-[3-(methylsulfonyl)phenyl]-1-propylpiperidine hydrochloride [(-)-OSU6162], and amitriptyline. Dextromethorphan and MAMC O-dealkylation activity in buffered carbonate was approximately 25 and 38%, respectively, relative to phosphate, while activity in sulfate and acetate buffers displayed minor differences. In contrast, N-dealkylation reactions for both (-)-OSU6162 and amitriptyline were unaffected by the presence of carbonate, and the other anions tested. Subsequent kinetic studies revealed that the basis of reduced turnover of dextromethorphan was primarily a V(max) effect, as the V(max) for the rate was 16.9 and 5.6 pmol/min/pmol P450 in phosphate and carbonate, respectively. Interestingly, similar rates of dextromethorphan O-demethylation in phosphate and carbonate were observed when reactions were supported by cumene hydroperoxide (CuOOH). Furthermore, it was observed that while CuOOH could equally support dextromethorphan O-demethylation compared to NADPH, amitriptyline N-demethylation was only minimally supported. Finally, intramolecular kinetic isotope effect (KIE) experiments with amitriptyline-d3 in CuOOH-supported reactions yielded a k(H)/k(D) of 5.2, substantially higher than in phosphate and carbonate supported by NADPH (k(H)/k(D)=1.5). Overall, results suggest that carbonate disrupts the relative ratios of the potential P450 oxygenating species, which differentially catalyze O- and N-dealkylation reactions mediated by CYP2D6.     

Unusual features for zirconium(IV) binding to human serum transferrin

Human serum apotransferrin (hTF) binds to Zr(IV) slowly in the presence of nitrilotriacetate (NTA), citrate or ethylenediaminetetraacetate (EDTA) as donor ligands. For Zr(NTA)(2)(2-) as donor, equilibrium was reached in ca. 2 h (pH 7.4, 298 K, 10 mM Hepes, 5 mM bicarbonate) and full loading of the N- and C-lobe sites was achievable to give Zr(2)-hTF. (13)C NMR data suggest that carbonate can bind as a synergistic anion. (1)H and 2D [(1)H,(13)C] (using epsilon-[(13)C]Met-hTF) NMR studies show that there is little lobe-selectively in the order of Zr(IV) uptake. Fe(III) displaced Zr(IV) from the C-lobe of Zr(2)-hTF first, followed by the N-lobe. However, in the presence of a large excess of NTA, Zr(IV) binds to the N-lobe of holo-hTF (Fe(2)-hTF) first followed by the C-lobe. The (1)H and (13)C NMR chemical shift changes for epsilon-[(13)CH(3)] of Met464, which is close to the C-lobe site, are quite distinct from those observed previously for Al(III), Fe(III), Ti(IV), Ga(III) and Bi(III) binding to hTF, suggesting that Zr(IV) binding may not induce lobe closure [as observed previously for Hf(IV)]. This may affect receptor recognition and play a role in the different biological behaviour of Zr(IV) compared to Ti(IV).     

Crystal structure at 1.9 A resolution of the apoovotransferrin N-lobe bound by sulfate anions: implications for the domain opening and iron release mechanism

Several lines of functional evidence have shown that anion binding to a nonsynergistic site is a prerequisite for the anion-mediated iron release mechanism of transferrins. We report here structural evidence of the location of sulfate anion binding sites of the ovotransferrin N-lobe via the 1.90 A resolution apo crystal structure. The crystals were grown in an ammonium sulfate solution and belonged to space group P6(3)22 with the following unit cell dimensions: a = b = 125.17 A and c = 87.26 A. The structural determination was performed by isomorphous replacement, using Pt and Au derivatives. The structure refinement gave an R-factor of 0.187 in the resolution range of 7.0-1.90 A for the final model. From the electron density map, the existence of four bound SO(4)(2)(-) anions was detected. Three of them that exhibited reasonably low B-factors were all located in the opened interdomain cleft (sites 1-3). In site 1, the bound anion directly interacts with an Fe(3+)-coordinating ligand; SO(4) O1 and SO(4) O3 form hydrogen bonds with His250 NE2. Oxygen atom O3 of the same sulfate anion makes a hydrogen bond with Ser91 OG in a hinge strand. The sulfate anion in site 2 partially occupies the synergistic anion binding sites; SO(4) O2 and SO(4) O3 are hydrogen bonded to Arg121 NE and NH2, respectively, both of which are consensus anchor groups for CO(3)(2)(-) anion in holotransferrins. The former oxygen atom of SO(4)(2)(-) is also hydrogen bonded to Ser122 N, which forms a hydrogen bond with Fe(3+)-coordinating ligand Asp60 OD2 in holotransferrins. Some of the SO(4)(2)(-) oxygen atoms in sites 1 and 2 interact indirectly through H(2)O molecules with functionally important protein groups, such as the other Fe(3+)-coordinating ligands, Tyr92 OH and Tyr191 OH, and a dilysine trigger group, Lys209 NZ. In site 3, SO(4) O1 and SO(4) O4 form hydrogen bonds with Ser192 OG and Tyr191 N, respectively, and SO(4) O2 forms hydrogen bonds with Ser192 N and Ser192 OG. These structural data are consistent with the view that the anion bindings to the interdomain cleft, especially to sites 1 and 2, play crucial roles in the domain opening and synergistic carbonate anion release in the iron release mechanism of the ovotransferrin N-lobe.     

A pulse-radiolysis study of the reaction of hydrated electrons with N'-formylkynurenine and related compounds: electron-transfer reactions with nucleic acid components.

The reactivity of N'-formylkynurenine (FK) derivatives towards eaq has been investigated. The reduced transient species have been characterized (lambda max approximately 340, 440 nm, epsilon lambda max approximately 3000-1000 M-1 cm-1, pKa approximately 7.8). Owing to the strong FK electron affinity, electron-transfer reactions occur from purine (except guanine) and pyrimidine electron adducts to FK (k approximately 2-7 x 10(9) M-1 s-1). As some FK derivatives bind to DNA (or polynucleotides) the protective effect of complexation on FK-DNA (or polynucleotides) adduct formation has been investigated.