Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid.

Autism spectrum disorders (ASD) are characterized by altered sociability, compromised communication and stereotyped/repetitive behaviors, for which no specific treatments are currently available. Prenatal exposure to valproic acid (VPA) is a known, although still underestimated, environmental risk factor for ASD. Altered endocannabinoid activity has been observed in autistic patients, and endocannabinoids are known to modulate behavioral traits that are typically affected in ASD. On this basis, we tested the hypothesis that changes in the endocannabinoid tone contribute to the altered phenotype induced by prenatal VPA exposure in rats, with focus on behavioral features that resemble the core and associated symptoms of ASD. In the course of development, VPA-exposed rats showed early deficits in social communication and discrimination, compromised sociability and social play behavior, stereotypies and increased anxiety, thus providing preclinical proof of the long-lasting deleterious effects induced by prenatal VPA exposure. At the neurochemical level, VPA-exposed rats displayed altered phosphorylation of CB1 cannabinoid receptors in different brain areas, associated with changes in anandamide metabolism from infancy to adulthood. Interestingly, enhancing anandamide signaling through inhibition of its degradation rescued the behavioral deficits displayed by VPA-exposed rats at infancy, adolescence and adulthood. This study therefore shows that abnormalities in anandamide activity may underlie the deleterious impact of environmental risk factors on ASD-relevant behaviors and that the endocannabinoid system may represent a therapeutic target for the core and associated symptoms displayed by autistic patients.

PML nuclear body disruption impairs DNA double-strand break sensing and repair in APL.

Proteins involved in DNA double-strand break (DSB) repair localize within the promyelocytic leukemia nuclear bodies (PML-NBs), whose disruption is at the root of the acute promyelocytic leukemia (APL) pathogenesis. All-trans-retinoic acid (RA) treatment induces PML-RARα degradation, restores PML-NB functions, and causes terminal cell differentiation of APL blasts. However, the precise role of the APL-associated PML-RARα oncoprotein and PML-NB integrity in the DSB response in APL leukemogenesis and tumor suppression is still lacking. Primary leukemia blasts isolated from APL patients showed high phosphorylation levels of H2AX (γ-H2AX), an initial DSBs sensor. By addressing the consequences of ionizing radiation (IR)-induced DSB response in primary APL blasts and RA-responsive and -resistant myeloid cell lines carrying endogenous or ectopically expressed PML-RARα, before and after treatment with RA, we found that the disruption of PML-NBs is associated with delayed DSB response, as revealed by the impaired kinetic of disappearance of γ-H2AX and 53BP1 foci and activation of ATM and of its substrates H2AX, NBN, and CHK2. The disruption of PML-NB integrity by PML-RARα also affects the IR-induced DSB response in a preleukemic mouse model of APL in vivo. We propose the oncoprotein-dependent PML-NB disruption and DDR impairment as relevant early events in APL tumorigenesis.

Heme-based catalytic properties of human serum albumin.

Human serum albumin (HSA): (i) controls the plasma oncotic pressure, (ii) modulates fluid distribution between the body compartments, (iii) represents the depot and carrier of endogenous and exogenous compounds, (iv) increases the apparent solubility and lifetime of hydrophobic compounds, (v) affects pharmacokinetics of many drugs, (vi) inactivates toxic compounds, (vii) induces chemical modifications of some ligands, (viii) displays antioxidant properties, and (ix) shows enzymatic properties. Under physiological and pathological conditions, HSA has a pivotal role in heme scavenging transferring the metal-macrocycle from high- and low-density lipoproteins to hemopexin, thus acquiring globin-like reactivity. Here, the heme-based catalytic properties of HSA are reviewed and the structural bases of drug-dependent allosteric regulation are highlighted.

Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells.

Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17ß-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.

Neuroglobin upregulation induced by 17ß-estradiol sequesters cytocrome c in the mitochondria preventing H2O2-induced apoptosis of neuroblastoma cells.

The sex steroid hormone 17ß-estradiol (E2) upregulates the levels of neuroglobin (NGB), a new neuroprotectant globin, to elicit its neuroprotective effect against H(2)O(2)-induced apoptosis. Several mechanisms could be proposed to justify the NGB involvement in E2 prevention of stress-induced apoptotic cell death. Here, we evaluate the ability of E2 to modulate the intracellular NGB localization and the NGB interaction with mitochondrial cytochrome c following the H(2)O(2)-induced toxicity. Present results demonstrate that NGB is expressed in the nuclei, mitochondria, and cytosol of human neuroblastoma SK-N-BE cells. E2, but not H(2)O(2) treatment of SK-N-BE cells, reallocates NGB mainly at the mitochondria and contemporarily reduces the number of apoptotic nuclei and the levels of cleaved caspase-3. Remarkably, the E2 treatment strongly increases NGB-cytochrome c association into mitochondria and reduces the levels of cytochrome c into the cytosol of SK-N-BE cells. Although both estrogen receptors (ERα and ERß) are expressed in the nucleus, mitochondria, and cytosol of SK-N-BE cells, this E2 effect specifically requires the mitochondrial ERß activity. As a whole, these data demonstrate that the interception of the intrinsic apoptotic pathway into mitochondria (i.e., the prevention of cytochrome c release) is one of the pivotal mechanisms underlying E2-dependent NGB neuroprotection against H(2)O(2) toxicity.

17ß-Oestradiol anti-inflammatory effects in primary astrocytes require oestrogen receptor ß-mediated neuroglobin up-regulation.

Neuroglobin (Ngb), so named after its initial discovery in brain neurones, has received great attention as a result of its neuroprotective effects both in vitro and in vivo. Recently, we demonstrated that, in neurones, Ngb is a 17ß-oestradiol (E(2) ) inducible protein that is pivotal for hormone-induced anti-apoptotic effects against H(2) O(2) toxicity. The involvement of Ngb in other brain cell populations, as well as in other neuroprotective effects of E(2) , is completely unknown at present. We demonstrate Ngb immunoreactivity in reactive astrocytes located in the proximity of a penetrating cortical injury in vivo and the involvement of Ngb in the E(2) -mediated anti-inflammatory effect in primary cortical astrocytes. Upon binding to oestrogen receptor (ER)ß, E(2) enhances Ngb levels in a dose-dependent manner. Although with a lesser degree than E(2) , the pro-inflammatory stimulation with lipopolysaccharide (LPS) also induces the increase of Ngb protein levels via nuclear factor-(NF)κB signal(s). Moreover, a negative cross-talk between ER subtypes and NFκB signal(s) has been demonstrated. In particular, ERα-activated signals prevent the NFκB-mediated Ngb increase, whereas LPS impairs the ERß-induced up-regulation of Ngb. Therefore, the co-expression of both ERα and ERß is pivotal for mediating E(2) -induced Ngb expression in the presence of NFκB-activated signals. Interestingly, Ngb silencing prevents the effect of E(2) on the expression of inflammatory markers (i.e. interleukin 6 and interferon γ-inducible protein 10). Ngb can be regarded as a key mediator of the different protective effects of E(2) in the brain, including protection against oxidative stress and the control of inflammation, both of which are at the root of several neurodegenerative diseases.

A neglected giant subcutaneous leiomyosarcoma of the shoulder: a case report.

Superficial leiomyosarcoma is an exceeding uncommon malignant tumor, which can be located either cutaneous or subcutaneous tissues. It may occur anywhere in the body, but there is a predilection for the thighs. The distinction between cutaneous or subcutaneous, increased mass size (> 5 cm) and depth are considered to have worst prognosis in terms of recurrence and metastasis. We report a rare case of an 81-year-old woman, presented with a 20-cm leiomyosarcoma of the shoulder. The patient reported that the mass was completely asymptomatic and its growth was slow. A CT study showed a lesion closely apposed to the head of the humerus and revealed no a clear relationship with the surrounding muscles. There was no regional lymphadenopathy or the evidence of metastatic disease. After tumor resection, a large skin and soft-tissue defect was remained and the repair was performed by myocutaneous rotational free flap of latissimus dorsi. The conclusion of the histological analysis was: subcutaneous leiomyosarcoma pT2--grade 1+ (NCI system). The delayed diagnosis and the surgical treatment and giant size of soft tissue leiomyosarcoma may adversely affect the final result.

Human haptoglobin structure and function--a molecular modelling study.

Hemoglobin is the most prominent protein in blood, transporting O(2) and facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin metabolism leads to the release of extra-erythrocytic hemoglobin, with potentially severe consequences for health. Extra-erythrocytic hemoglobin is complexed to haptoglobin for clearance by tissue macrophages. The human gene for haptoglobin consists of three structural alleles: Hp1F, Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only one amino acid, whereas the Hp2 allele is the result of a fusion of the Hp1F and Hp1S alleles, is present only in humans and gives rise to a longer alpha-chain. Haptoglobin consists of a dimer of alphabeta-chains covalently linked by a disulphide bond between the Cys15 residue of each alpha-chain. However, the presence of the Hp1 and Hp2 alleles in humans gives rise to HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two free Cys residues (Cys15 and Cys74) whose participation in intermolecular disulphide bonds gives rise to higher-order covalent multimers. Here, the complete modelling of both haptoglobin variants, together with their basic quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is reported. The structural details of the models, which represent the first complete view of the molecular details of human haptoglobin variants, are discussed in relation to the known haptoglobin function(s).

A novel multicompartimental system based on aminated poly(vinyl alcohol) microspheres/succinoylated pullulan microspheres for oral delivery of anionic drugs.

Poly(vinyl alcohol) (PVA) microspheres were prepared by dispersion reticulation with glutaraldehyde and further aminated. These microspheres were firstly loaded with diclofenac (DF) and then entrapped in cellulose acetate butyrate (CAB) microcapsules by an o/w solvent evaporation technique for intestinal delivery of drug. The encapsulated PVA microspheres due to their low swelling degree in intestinal fluids, do not have enough force to produce the disruption of CAB shell, therefore different amounts of succinoylated pullulan microspheres (SP-Ms) (exchange capacity up to 5.2 meq/g) were co-encapsulated. The SP-Ms do not swell in acidic pH, but swell up to 20-times in intestinal fluids causing the rupture of CAB shell and facilitating the escape of loaded PVA microspheres.

Nitric oxide impairs the 17beta-estradiol-induced apoptosis in human colon adenocarcinoma cells.

Nitric oxide (NO) and 17beta-estradiol (E2) are both important in gastrointestinal health and disease. NO contributes to gastrointestinal motility as well as to inflammation and carcinogenic processes. By contrast, E2 reduces the incidence of colon adenoma and carcinoma by about 30%. We report the genomic and non-genomic E2-estrogen receptor (ER) beta-induced effects in human colon adenocarcinoma. The effect of NO on ERbeta activities was also assessed. The E2-ERbeta-dependent gene transcription was inhibited by exogenous NO, whereas some non-genomic E2-dependent effects (e.g. p38/MAP kinase), important for the activation of the apoptotic cascade, were unaffected by NO. However, NO impaired the E2-induced pro-apoptotic cascade in human colon adenocarcinoma cells by inhibiting caspase-3. The effects of NO may reflect chemical modification(s) of Cys residues present in the DNA recognition domain of ERbeta as well as in the caspase-3 active site. On the whole, high NO concentrations suppressed the E2 protective effects in the gastrointestinal tract, suggesting that the caspase-dependent apoptotic cascade may become critical under conditions of high redox stress such as occur under specific activation of the immune system by chronic infections or pathogen challenge.

[Nitric oxide and anti-protozoan chemotherapy]. / L'ossido nitrico nella terapia anti-protozoaria.

Constitutive nitric oxide (NO) is generated by constitutively expressed types of NO-synthase enzymes (NOS-I and -III), being involved in physiological processes such as nervous transmission and vasodilatation. Inducible NO, synthesized by the NO-synthase isoform NOS-II, is an anti-pathogen and tumoricidal agent. However, inducible NO production requires a tight control because of cytotoxic and immune-modulation activity. NO produced by human and canine macrophages has long been demonstrated to be involved in the intracellular killing of Leishmania. Mechanisms of parasite survival and persistence in the host have been throughly investigated, and include suppression of NOS-II and the parasite entry into NOS-II negative cells. Both intracellular and extracellular morphotypes of Trypanosoma cruzi are killed by NO in vitro and in vivo, although a role of NO in the pathogenesis of heart disease has been reported. Killing of extracellular protozoa such as Trichomonas vaginalis and Naegleria fowleri by activated macrophages is also mediated by NO. The main control of Plasmodium spp infection in human and murine hepatocytes, and in human monocytes is achieved by NO-mediated mechanisms. Protection from severe malaria in African children has been found associated with polymorphisms of the NOS-II promoter; however, a pathogenic role of endogenous NO has been documented in cerebral malaria. Although several macromolecules are putative NO targets, recent experimental work has shown that NO-releasing compounds inhibit cysteine proteases (CP) of P. falciparum, T. cruzi and L. infantum in a dose-dependent manner. CPs are present in a wide range of parasitic protozoa and appear to be relevant in several aspects of the life cycle and of the parasite-host relationships. Comparative analysis of 3-D amino acid sequence models of CPs from a broad range of living organisms, from viruses to mammals, suggests that the Sy atom of the Cys catalytic residue undergoes NO-dependent chemical modification (S-nytrosilation and disulfide bridge formation), with the concomitant loss of enzyme activity. The NO-donor S-nitroso-N-acetilpenicillamine (SNAP) was shown to kill T. cruzi epimastigotes and L. infantum promastigotes in culture, while a combination of nitrite plus acid organic salts was highly effective against L. major amastigotes in mouse macrophages. A parasitostatic effect--with both encystation and excystation inhibition--of S-nitrosoglutathione and spermine-NONOate was documented in trophozoite cultures of Giardia duodenalis. Recently, a novel formulation of metronidazole bearing a NO-releasing group was found to enhance significantly the in vitro killing of Entamoeba histolytica trophozoites, compared to metronidazole. So far, only two clinical studies were performed on human patients, suffering from cutaneous leishmaniasis. In one study, 16 Ecuadorean patients were treated with a SNAP cream administered on lesions for 10 days. All lesions were parasitologically cured and clinically healed by day 30. In the second study, a different NO-producing cream (basically nitrite in acidic environment) was employed to treat 40 Syrian patients. Only 28% of them showed improvement and 12% were cured by day 60. In conclusion, despite the wide evidence that NO can be regarded as a natural anti-protozoal weapon, little efforts have been made to develop and test NO-based drugs in human medicine. This is mainly due to the difficulty in designing suitable chemical carriers able to release the right amount of NO, in the right place and in the right time, to avoid toxic effects against non-target host cells.

Effect of ibuprofen and warfarin on the allosteric properties of haem-human serum albumin. A spectroscopic study.

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem-human serum albumin (ferric HSA-haem) and of ferrous nitrosylated haem-human serum albumin (ferrous HSA-haem-NO) is reported. Ferric HSA-haem is hexa-coordinated, the haem-iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules--buried in close proximity to the haem binding pocket--induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem-iron, and the quenching of the 1H-NMR relaxivity. Values of Kd for ibuprofen and warfarin binding to the warfarin primary site of ferric HSA-haem, corresponding to the ibuprofen secondary cleft, are 5.4 +/- 1.1 x 10(-4) m and 2.1 +/- 0.4 x 10(-5) m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA-haem is lower than that reported for drug binding to haem-free HSA. Accordingly, the Kd value for haem binding to HSA increases from 1.3 +/- 0.2 x 10(-8) m in the absence of drugs to 1.5 +/- 0.2 x 10(-7) m in the presence of ibuprofen and warfarin. Ferrous HSA-haem-NO is a five-coordinated haem-iron system. Drug binding to the warfarin primary site of ferrous HSA-haem-NO induces the transition towards the six-coordinated haem-iron species, the haem-iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear-cut evidence for the drug-induced shift of allosteric equilibrium(a) of HSA.

Contryphan-Vn: a novel peptide from the venom of the Mediterranean snail Conus ventricosus.

The isolation, purification, and biochemical characterization of the novel peptide Contryphan-Vn, extracted from the venom of the Mediterranean marine snail Conus ventricosus, is reported. Contryphan-Vn is the first Conus peptide described from a vermivorous species and the first purified from the venom of the single Mediterranean Conus species. The amino acid sequence of Contryphan-Vn is As with other contryphans, Contryphan-Vn contains a d-tryptophan residue, is amidated at the C-terminus, and maintains the five-residue intercystine loop size. However, Contryphan-Vn differs from the known contryphans by the insertion of the Asp residue at position 2, by the lack of hydroxylation of Pro(4), and, remarkably, by the presence of the basic residue Lys(6) within the intercystine loop. Although the biological function(s) of contryphans is still unknown, these characteristics suggest distinct molecular target(s) and/or function(s) for Contryphan-Vn.

Inhibition of Saccharomyces cerevisiae phosphomannose isomerase by the NO-donor S-nitroso-acetyl-penicillamine.

Phosphomannose isomerase (PMI; EC. 5.3.1.8) is an essential metalloenzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. The Cys150 residue (according to Candida albicans PMI numbering) is conserved in the active centre of mammalian and yeast PMI, but not in bacterial species where it is replaced by Asn. Here, the dose- and time-dependent inhibitory effect of the NO-donor S-nitroso-acetyl-penicillamine on the Saccharomyces cerevisiae PMI catalytic activity is reported. The analysis of the X-ray crystal structure of C. albicans PMI and of the molecular model of S. cerevisiae PMI provides a rationale for the low reactivity of Cys150 towards alkylating and nitrosylating agents.

Crystallization and preliminary X-ray analysis of neural haemoglobin from the nemertean worm Cerebratulus lacteus.

The nemertean worm Cerebratulus lacteus neural tissue haemoglobin (109 amino acids, the shortest known haemoglobin) has been overexpressed in Escherichia coli, purified and crystallized. A highly redundant native data set has been collected at the Cu K(alpha) wavelength to 2.05 A resolution. The crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with unit-cell parameters a = 42.5, b = 43.1, c = 60.2 A and one molecule per asymmetric unit. The anomalous difference Patterson map clearly reveals the position of the haem Fe atom, thus paving the way for MAD/SAD structure determination.

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme.

Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.

Nitric oxide inhibits selectively the 17beta-estradiol-induced gene expression without affecting nongenomic events in HeLa cells.

17beta-Estradiol (E2) induces genomic (i.e., pC3-luciferase promoter-reporter construct expression) and nongenomic (i.e., DNA synthesis and IP(3) production) effects in HeLa cells only after transient transfection with the human estrogen receptor alpha (ERalpha) reporter plasmid. Here the effect of nitric oxide (NO) on both E2-induced effects in transiently transfected HeLa cells is reported. Remarkably, the E2-dependent gene transcription is inhibited dose-dependently by NO. By contrast, DNA synthesis and IP(3) production, representing nongenomic E2-dependent effects, are unaffected by NO. The selective NO action on E2-induced functions may be related to NO-mediated chemical modification(s) of the Cys residues present in the DNA recognition domain of ERalpha impairing DNA binding.

Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin.

The monomer-dimer equilibrium and the oxygen binding properties of ferrous recombinant Vitreoscilla hemoglobin (Vitreoscilla Hb) have been investigated. Sedimentation equilibrium data indicate that the ferrous deoxygenated and carbonylated derivatives display low values of equilibrium dimerization constants, 6 x 10(2) and 1 x 10(2) M(-1), respectively, at pH 7.0 and 10 degrees C. The behavior of the oxygenated species, as measured in sedimentation velocity experiments, is superimposable to that of the carbonylated derivative. The kinetics of O(2) combination, measured by laser photolysis at pH 7.0 and 20 degrees C, is characterized by a second-order rate constant of 2 x 10(8) M(-1) s(-1) whereas the kinetics of O(2) release at pH 7.0 is biphasic between 10 and 40 degrees C, becoming essentially monophasic below 10 degrees C. Values of the first-order rate constants (at 20 degrees C) and of the activation energies for the fast and slow phases of the Vitreoscilla Hb deoxygenation process are 4.2 s(-1) and 19.2 kcal mol(-1) and 0.15 s(-1) and 24.8 kcal mol(-1), respectively. Thus the biphasic kinetics of Vitreoscilla Hb deoxygenation is unrelated to the association state of the protein. The observed biphasic oxygen release may be accounted for by the presence of two different conformers in thermal equilibrium within the monomer. The two conformers may be assigned to a structure in which the heme-iron-bound ligand is stabilized by direct hydrogen bonding to TyrB10 and a structure in which such interaction is absent. The slow interconversion between the two conformers may reflect a very large conformational rearrangement in the disordered distal pocket segment connecting helices C and E.

Relaxometric characterization of human hemalbumin.

Hemalbumin [i.e., Fe(III)-protoporphyrin IX-human serum albumin; Fe(III)heme-HSA] is an important intermediate in the recovery of heme iron following hemolysis. Relaxometric data are consistent with the occurrence of a hexacoordinated high-spin Fe(III) center with no water in the inner coordination sphere. The relatively high relaxation enhancement observed for an aqueous solution of Fe(III)heme-HSA (r1p=4.8 mM(-1)s(-1) at 20 MHz, pH 7, and 25 C) is ascribed to the occurrence of a strong contribution from water molecules in the second coordination sphere. Structural analysis of the putative binding region has been performed by a Monte Carlo simulated annealing procedure, which allowed us to identify His105 and Tyr148 as axial ligands. The role of a tyrosinate as the sixth Fe(III)heme ligand is supported by the pH-dependent analysis. Interestingly, when Fe(III) is replaced by Mn(III), the occurrence of a fast exchanging water molecule at pH values close to neutrality is detected. As the pH is increased, the Mn(III) containing system behaves analogously to Fe(III)heme-HSA. At higher pH, the phenolate ligand is eventually displaced by OH- from both Fe(III) and Mn(III) centers. Support for the proposed bonding scheme has been gained also from competitive binding assays for the sixth coordination site by fluoride, azide, and imidazole ligands.