Protoporphyrin IX iron(II) revisited. An overview of the Mössbauer spectroscopic parameters of low-spin porphyrin iron(II) complexes.

Mössbauer parameters of low-spin six-coordinate [Fe(II)(Por)L2] complexes (where Por is a synthetic porphyrin; L is a nitrogenous aliphatic, an aromatic base or a heterocyclic ligand, a P-bonding ligand, CO or CN) and low-spin [Fe(Por)LX] complexes (where L and X are different ligands) are reported. A known point charge calculation approach was extended to investigate how the axial ligands and the four porphyrinato-N atoms generate the observed quadrupole splittings (ΔEQ) for the complexes. Partial quadrupole splitting (p.q.s.) and partial chemical shifts (p.c.s.) values were derived for all the axial ligands, and porphyrins reported in the literature. The values for each porphyrin are different emphasising the importance/uniqueness of the [Fe(PPIX)] moiety, (which is ubiquitous in nature). This new analysis enabled the construction of figures relating p.c.s and p.q.s values. The relationships presented in the figures indicates that strong field ligands such as CO can, and do change the sign of the electric field gradient in the [Fe(II)(Por)L2] complexes. The limiting p.q.s. value a ligand can have and still form a six-coordinate low-spin [Fe(II)(Por)L2] complex is established. It is shown that the control the porphyrin ligands exert on the low-spin Fe(II) atom limits its bonding to a defined range of axial ligands; outside this range the spin state of the iron is unstable and five-coordinate high-spin complexes are favoured. Amongst many conclusions, it was found that oxygen cannot form a stable low-spin [Fe(II)(Por)L(O2)] complex and that oxy-haemoglobin is best described as an [Fe(III)(Por)L(O2-)] complex, the iron is ferric bound to the superoxide molecule.

The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core.

Ferritins are multimeric cage-forming proteins that play a crucial role in cellular iron homeostasis. All H-chain-type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α-helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron-release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme-ferroxidase centre electron transfer pathway.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c'-ß: Implications for NO sensors.

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-ß) resembling that of cytochromes P460. A recent structure of cyt c'-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations.

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions. Under hypoxia, cytoglobin generates nitric oxide, which is strongly modulated by the oxidation state of the cysteines. This gives a plausible role for this biochemistry in controlling nitric oxide homeostasis. Mutations to control specific properties of hemoglobin and myoglobin, including nitric oxide binding/scavenging and the nitrite reductase activity of various globins, have been reported. We have mapped these key mutations onto cytoglobin, which represents the E7 distal ligand, B2/E9 disulfide, and B10 heme pocket residues, and examined the nitric oxide binding, nitric oxide dioxygenase activity, and nitrite reductase activity. The Leu46Trp mutation decreases the nitric oxide dioxygenase activity > 10,000-fold over wild type, an effect 1000 times greater than similar mutations with other globins. By understanding how particular mutations can affect specific reactivities, these mutations may be used to target specific cytoglobin activities in cell or animal models to help understand the precise role(s) of cytoglobin under physiological and pathophysiological conditions.

Studies on the binding of nitrogenous bases to protoporphyrin IX iron(II) in aqueous solution at high pH values.

Studies are reported on the formation of low-spin six-coordinate [Fe(PPIX)L2] complexes from iron(II) protoporphyrin where L is one of a series of nitrogenous ligands (aliphatic, aromatic or heterocyclic). The bonding constants have been determined by titration of the metal complex with these ligands and are compared in relation to previous studies. The adduct formation was monitored utilising optical spectroscopy. In addition, MÓ§ssbauer spectroscopic experiments were conducted to monitor the electronic environment around the central iron atom in these complexes. The two complementary spectroscopic methods indicated that all nitrogen ligands formed low-spin octahedral complexes. The magnitude of the overall binding constants (ß2 values) are discussed and related to (a) the pKa values of the free ligands and (b) the Mössbauer parameter ΔEQ, which represents the quadrupole splitting of the haem iron. The ß2 and ΔEQ values are also discussed in terms of the structure of the ligand. Cooperative binding was observed for nearly all the ligands with Hill coefficients close to 2 for iron(II) protoporphyrin; one of these ligands displayed a much greater affinity than any we previously studied, and this was a direct consequence of the structure of the ligand. Overall conclusions on these and previous studies are drawn in terms of aliphatic ligands versus aromatic ring structures and the absence or presence of sterically hindered nitrogen atoms. The implications of the work for the greater understanding of haem proteins in general and in particular how the nitrogenous ligand binding results are relevant to and aid the understanding of the binding of inhibitor molecules to the cytochrome P450 mono-oxygenases (for therapeutic purposes) are also discussed. Changes in the electronic absorption spectra of five-coordinate [Fe(II)(PPIX)(2-MeIm)] that occurred as the temperature was lowered from room temperature to 78° K.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2 O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+ , was exposed to O2 or H2 O2 . We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2 O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O2 . Initially formed Fe3+ can further react with H2 O2 (producing protein bound radicals) but relaxes within seconds to an H2 O2 -unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2 O2 rather than sequester iron.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.

Bacterial iron detoxification at the molecular level.

Iron is an essential micronutrient, and, in the case of bacteria, its availability is commonly a growth-limiting factor. However, correct functioning of cells requires that the labile pool of chelatable "free" iron be tightly regulated. Correct metalation of proteins requiring iron as a cofactor demands that such a readily accessible source of iron exist, but overaccumulation results in an oxidative burden that, if unchecked, would lead to cell death. The toxicity of iron stems from its potential to catalyze formation of reactive oxygen species that, in addition to causing damage to biological molecules, can also lead to the formation of reactive nitrogen species. To avoid iron-mediated oxidative stress, bacteria utilize iron-dependent global regulators to sense the iron status of the cell and regulate the expression of proteins involved in the acquisition, storage, and efflux of iron accordingly. Here, we survey the current understanding of the structure and mechanism of the important members of each of these classes of protein. Diversity in the details of iron homeostasis mechanisms reflect the differing nutritional stresses resulting from the wide variety of ecological niches that bacteria inhabit. However, in this review, we seek to highlight the similarities of iron homeostasis between different bacteria, while acknowledging important variations. In this way, we hope to illustrate how bacteria have evolved common approaches to overcome the dual problems of the insolubility and potential toxicity of iron.

A Histidine Residue and a Tetranuclear Cuprous-thiolate Cluster Dominate the Copper Loading Landscape of a Copper Storage Protein from Streptomyces lividans.

The chemical basis for protecting organisms against the toxic effect imposed by excess cuprous ions is to constrain this through high-affinity binding sites that use cuprous-thiolate coordination chemistry. In bacteria, a family of cysteine rich four-helix bundle proteins utilise thiolate chemistry to bind up to 80 cuprous ions. These proteins have been termed copper storage proteins (Csp). The present study investigates cuprous ion loading to the Csp from Streptomyces lividans (SlCsp) using a combination of X-ray crystallography, site-directed mutagenesis and stopped-flow reaction kinetics with either aquatic cuprous ions or a chelating donor. We illustrate that at low cuprous ion concentrations, copper is loaded exclusively into an outer core region of SlCsp via one end of the four-helix bundle, facilitated by a set of three histidine residues. X-ray crystallography reveals the existence of polynuclear cuprous-thiolate clusters culminating in the assembly of a tetranuclear [Cu4 (µ2 -S-Cys)4 (Νδ1 -His)] cluster in the outer core. As more cuprous ions are loaded, the cysteine lined inner core of SlCsp fills with cuprous ions but in a fluxional and dynamic manner with no evidence for the assembly of further intermediate polynuclear cuprous-thiolate clusters as observed in the outer core. Using site-directed mutagenesis a key role for His107 in the efficient loading of cuprous ions from a donor is established. A model of copper loading to SlCsp is proposed and discussed.

A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P1-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

Active-site maturation and activity of the copper-radical oxidase GlxA are governed by a tryptophan residue.

GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best characterised. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic sites in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper-co-ordinating tyrosine ligand and houses a radical. In GlxA and Gox, a second co-ordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes, creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here, we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.

Copper and nickel bind via two distinct kinetic mechanisms to a CsoR metalloregulator.

The intricate interplay between polypeptide and metal ion binding underscores many of life's fundamental processes. Metalloregulators recognise and bind cognate metal ions during cellular metal stress, evoking a transcriptional response so as to maintain metal ion homeostasis. Members of the copper sensitive operon repressor (CsoR) family of metalloregulators bind to their operator DNA in the absence of a bound metal ion, but on binding Cu(I) an allosteric conformational switch is induced that causes dissociation of the bound DNA. Other divalent metal ions are capable of binding to CsoR members but do not induce the allosteric response observed with Cu(I). The thermodynamics of Cu(I) binding has been studied in this family of metalloregulators, but the binding kinetics and mechanism of Cu(I) or a non-cognate metal ion is unknown. In the present study we have used stopped-flow absorbance kinetics and site-directed variants of the CsoR from Streptomyces lividans to monitor binding of Cu(I) and non-cognate Ni(II). The variants have been designed to individually replace known metal ion binding ligands and also to test the role of a histidine residue (His103) close, but not considered part of the Cu(I) first coordination sphere. Cu(I)/Ni(II) ion displacement studies have also been investigated. The kinetic data are most consistent with the existence of two distinct mechanisms that account for Cu(I) and Ni(II) ion binding to this CsoR. In particular Ni(II) has two binding sites; one that has identical amino acid coordination as the Cu(I) binding site and the second involving His103, a residue determined here not to be involved in the mechanism of Cu(I) binding.

Sickle Cell Hemoglobin in the Ferryl State Promotes ßCys-93 Oxidation and Mitochondrial Dysfunction in Epithelial Lung Cells (E10).

Polymerization of intraerythrocytic deoxyhemoglobin S (HbS) is the primary molecular event that leads to hemolytic anemia in sickle cell disease (SCD). We reasoned that HbS may contribute to the complex pathophysiology of SCD in part due to its pseudoperoxidase activity. We compared oxidation reactions and the turnover of oxidation intermediates of purified human HbS and HbA. Hydrogen peroxide (H2O2) drives a catalytic cycle that includes the following three distinct steps: 1) initial oxidation of ferrous (oxy) to ferryl Hb; 2) autoreduction of the ferryl intermediate to ferric (metHb); and 3) reaction of metHb with an additional H2O2 molecule to regenerate the ferryl intermediate. Ferrous and ferric forms of both proteins underwent initial oxidation to the ferryl heme in the presence of H2O2 at equal rates. However, the rate of autoreduction of ferryl to the ferric form was slower in the HbS solutions. Using quantitative mass spectrometry and the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide, we found more irreversibly oxidized ßCys-93in HbS than in HbA. Incubation of the ferric or ferryl HbS with cultured lung epithelial cells (E10) induced a drop in mitochondrial oxygen consumption rate and impairment of cellular bioenergetics that was related to the redox state of the iron. Ferryl HbS induced a substantial drop in the mitochondrial transmembrane potential and increases in cytosolic heme oxygenase (HO-1) expression and mitochondrial colocalization in E10 cells. Thus, highly oxidizing ferryl Hb and heme, the product of oxidation, may be central to the evolution of vasculopathy in SCD and may suggest therapeutic modalities that interrupt heme-mediated inflammation.

Coupling of disulfide bond and distal histidine dissociation in human ferrous cytoglobin regulates ligand binding.

Earlier kinetics studies on cytoglobin did not assign functional properties to specific structural forms. Here, we used defined monomeric and dimeric forms and cysteine mutants to show that an intramolecular disulfide bond (C38-C83) alters the dissociation rate constant of the intrinsic histidine (H81) (∼1000 fold), thus controlling binding of extrinsic ligands. Through time-resolved spectra we have unequivocally assigned CO binding to hexa- and penta-coordinate forms and have made direct measurement of histidine rebinding following photolysis. We present a model that describes how the cysteine redox state of the monomer controls histidine dissociation rate constants and hence extrinsic ligand binding.

Cytoglobin ligand binding regulated by changing haem-co-ordination in response to intramolecular disulfide bond formation and lipid interaction.

Cytoglobin (Cygb) is a hexa-co-ordinate haem protein from the globin superfamily with a physiological function that is unclear. We have previously reported that the haem co-ordination is changed in the presence of lipids, potentially transforming the redox properties of the protein and hence the function of Cygb in vivo. Recent research suggests that the protein can exist in a number of states depending on the integrity and position of disulfide bonds. In the present study, we show that the monomeric protein with an internal disulfide bond between the two cysteine residues Cys38 and Cys83, interacts with lipids to induce a change in haem co-ordination. The dimeric protein with intermolecular disulfide bonds and monomeric protein without an intramolecular disulfide bond does not exhibit these changes in haem co-ordination. Furthermore, monomeric Cygb with an intramolecular disulfide bond has significantly different properties, oxidizing lipid membranes and binding ligands more rapidly as compared with the other forms of the protein. The redox state of these cysteine residues in vivo is therefore highly significant and may be a mechanism to modulate the biochemical properties of the haem under conditions of stress.

Dynamics of the heme-binding bacterial gas-sensing dissimilative nitrate respiration regulator (DNR) and activation barriers for ligand binding and escape.

DNR (dissimilative nitrate respiration regulator) is a heme-binding transcription factor that is involved in the regulation of denitrification in Pseudomonas aeruginosa. In the ferrous deoxy state, the heme is 6-coordinate; external NO and CO can replace an internal ligand. Using fluorescence anisotropy, we show that high-affinity sequence-specific DNA binding occurs only when the heme is nitrosylated, consistent with the proposed function of DNR as NO sensor and transcriptional activator. This role is moreover supported by the NO "trapping" properties revealed by ultrafast spectroscopy that are similar to those of other heme-based NO sensor proteins. Dissociated CO-heme pairs rebind in an essentially barrierless way. This process competes with migration out of the heme pocket. The latter process is thermally activated (Ea ∼ 7 kJ/mol). This result is compared with other heme proteins, including the homologous CO sensor/transcription factor CooA, variants of the 5-coordinate mycobacterial sensor DosT and the electron transfer protein cytochrome c. This comparison indicates that thermal activation of ligand escape from the heme pocket is specific for systems where an external ligand replaces an internal one. The origin of this finding and possible implications are discussed.

Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin.

A pathogenic V67M mutation occurs at the E11 helical position within the heme pockets of variant human fetal and adult hemoglobins (Hb). Subsequent post-translational modification of Met to Asp was reported in γ subunits of human fetal Hb Toms River (γ67(E11)Val → Met) and ß subunits of adult Hb (HbA) Bristol-Alesha (ß67(E11)Val → Met) that were associated with hemolytic anemia. Using kinetic, proteomic, and crystal structural analysis, we were able to show that the Met → Asp transformation involves heme cycling through its oxoferryl state in the recombinant versions of both proteins. The conversion to Met and Asp enhanced the spontaneous autoxidation of the mutants relative to wild-type HbA and human fetal Hb, and the levels of Asp were elevated with increasing levels of hydrogen peroxide (H2O2). Using H2(18)O2, we verified incorporation of (18)O into the Asp carboxyl side chain confirming the role of H2O2 in the oxidation of the Met side chain. Under similar experimental conditions, there was no conversion to Asp at the αMet(E11) position in the corresponding HbA Evans (α62(E11)Val → Met). The crystal structures of the three recombinant Met(E11) mutants revealed similar thioether side chain orientations. However, as in the solution experiments, autoxidation of the Hb mutant crystals leads to electron density maps indicative of Asp(E11) formation in ß subunits but not in α subunits. This novel post-translational modification highlights the nonequivalence of human Hb α, ß, and γ subunits with respect to redox reactivity and may have direct implications to α/ß hemoglobinopathies and design of oxidatively stable Hb-based oxygen therapeutics.

Haptoglobin binding stabilizes hemoglobin ferryl iron and the globin radical on tyrosine ß145.

AIM: Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection. RESULTS: Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit. INNOVATION: Hp binding decreases both the ferryl iron and free radical reactivity of Hb. CONCLUSION: Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging.