Trp180 of endothelial NOS and Trp56 of bacterial saNOS modulate sigma bonding of the axial cysteine to the heme

The proximal ligand of thiolate-coordinated heme proteins is crucial for the activation of the oxygen molecule and hydroxylation of substrates. In nitric oxide synthases (NOSs), the heme axial cysteine ligand forms a hydrogen bond to the side chain indole nitrogen of a tryptophan residue. Resonance Raman spectroscopy was used to probe W56F and W56Y variants of the NOS of Staphylococcus aureus (saNOS) and the analogous W180 variants of the endothelial NOS oxygenase domain (eNOSox). We show that the variants displayed lower ν_Fe-NO and ν_Fe-CO frequencies indicating that these mutations increased the electron density on the axial cysteine in their Fe^IIINO and Fe^IICO complexes. We also show by UV-visible spectroscopy that the Fe^IICO complexes of the variants displayed a red-shifted Soret optical transition in addition to the lower ν_Fe-CO thus establishing that these properties are sensitive indicators of the modulation of the basicity of the axial cysteine. We infer, based on its spectroscopic properties, that ferrous eNOSox W180Y saturated with l-arginine and tetrahydrobiopterin forms a tyrosine-cysteine hydrogen bond when bound to CO. Evidence for such a hydrogen bond was not obtained for the Fe^IIINO protein nor for the analogous saNOS variant. These mutations reveal interesting differences in the response of NOS isotypes to analogous mutations at conserved residues and clearly show that the heme-Fe to cysteine σ bond is modulated by the Cys-Trp hydrogen bond in NOSs. These studies serve as a basis to gain information on the role played by this hydrogen bond in oxygen activation in this class of enzymes.     

Disproportionation of hydroxylamine by water-soluble iron(III) porphyrinate compounds

The reactions of hydroxylamine (HA) with several water-soluble iron(III) porphyrinate compounds, namely iron(III) meso-tetrakis-(N-ethylpyridinium-2yl)-porphyrinate ([Fe^III(TEPyP)]⁵⁺), iron(III) meso-tetrakis-(4-sulphonatophenyl)-porphyrinate ([Fe^III(TPPS)]³⁻), and microperoxidase 11 ([Fe^III(MP11)]) were studied for different [Fe^III(Porph)]/[HA] ratios, under anaerobic conditions at neutral pH. Efficient catalytic processes leading to the disproportionation of HA by these iron(III) porphyrinates were evidenced for the first time. As a common feature, only N₂ and N₂O were found as gaseous, nitrogen-containing oxidation products, while NH₃ was the unique reduced species detected. Different N₂/N₂O ratios obtained with these three porphyrinates strongly suggest distinctive mechanistic scenarios: while [Fe^III(TEPyP)]⁵⁺ and [Fe^III(MP11)] formed unknown steady-state porphyrinic intermediates in the presence of HA, [Fe^III(TPPS)]³⁻ led to the well characterized soluble intermediate, [Fe^II(TPPS)NO]⁴⁻. Free-radical formation was only evidenced for [Fe^III(TEPyP)]⁵⁺, as a consequence of a metal centered reduction. We discuss the catalytic pathways of HA disproportionation on the basis of the distribution of gaseous products, free radicals formation, the nature of porphyrinic intermediates, the Fe^II/Fe^III redox potential, the coordinating capabilities of each complex, and the kinetic analysis. The absence of revealed either that no HAO-like activity was operative under our reaction conditions, or that , if formed, was consumed in the reaction milieu.     

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe^IIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe^IIINO dissociation rate (k_d) and NO association rate (k_on) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k_on and k_d by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O₂ catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O₂ intermediate, but instead were due to the slower NO k_on and k_d and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.     

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 × Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe⁺²) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe–S bond and yields five coordinate complexes. In the ferric (Fe⁺³) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe⁺³–NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe⁺²–NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.     

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of α1β1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain β1(1–385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (β1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat β1 distal heme pocket (Ile-145 → Tyr and Ile-149 → Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe^II-O₂ complexes and is likely the first observation of a Fe^II-O₂ complex in the full-length α1β1 protein. Additionally, these data suggest that atypical sGCs stabilize O₂ binding by a hydrogen bonding network involving tyrosine and glutamine.     

Nitric oxide binding to cationic and anionic ferric porphyrins in aqueous solution: reversible formation of ferrous NO species of the cationic porphyrin

Two water-soluble ferric porphyrins, sodium 5α,10β,15α,20β-tetrakis(2-(sulfonatoacetamido)phenyl)porphyrinatoiron(III) (Fe^IIITanP) and 5α,10β,15α,20β-tetrakis(2-(N,N,N-trimethylammoniumacetamido)phenyl)porphyrinatoiron(III) chloride (Fe^IIITcatP), were synthesized. The pK_a values of the coordinated H₂O of Fe^IIITanP and Fe^IIITcatP were evaluated to be 8.0 and 4.1, respectively. Reactions of NO with the ferric porphyrins were examined spectrophotometrically in aqueous solution. Porphyrin Fe^IIITanP binds NO reversibly to give the corresponding ferric NO species at pH 1.3 and pH 3.0, and Fe^IIITcatP reacts similarly with NO at pH 1.3. The thermodynamic data for the NO binding were estimated from van't Hoff plots. At pH 3.0, visible and ESR spectral data indicated that Fe^IIITcatP binds NO reversibly to produce ferrous NO species depending on NO partial pressures. These results were discussed based on through-space intramolecular interactions between the coordinated H₂O or NO and the ionic substituents of the porphyrins.     

Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

Synthesis and structural determination of a new five-coordinate iron(III) porphyrin containing monoanion 1,4-phenyldicyanamide as axial ligand

A new five coordinate and stable iron(III) heme analog, [Fe^III(OEP)(DicydH)], where OEP is the dianion of octaethylporphyrin and DicydH = monoanion of 1,4-phenyldicyanamide, has been synthesized. The compound has been characterized by different spectroscopic methods ¹H NMR, UV-Vis, IR as well as elemental analysis. ¹H NMR spectroscopy and magnetic moment measurements show that [Fe^III(OEP)(DicydH)] is paramagnetic and iron is five-coordinate. The structure of [Fe^III(OEP)(DicydH)] has been determined by X-ray diffraction analysis, that it is similar with a P2₁/c space group in the monoclinic crystal system. The crystal structure of the complex is stabilized by hydrogen bonds of the type N-H?N. Electrochemical of [Fe^III(OEP)(DicydH)] has been studied by cyclic voltammetry.     

The McClure and Weiss models of Fe–O2 bonding for oxyhemes, and the HbO2 + NO reaction

For the Fe–O₂(S = 0) linkages of oxyhemes, valence bond (VB) structures are re-presented for the McClure [Fe^II(S = 1) + O₂(S = 1)], Pauling–Coryell [Fe^II(S = 0) + O₂*(S = 0)], and Weiss [Fe^III(S = ½) + O₂ ^?(S = ½)] models of bonding. The VB structures for the McClure and Weiss models are of the increased-valence type, with more electrons participating in bonding than occur in their component Lewis structures. The Fe–O bond number and O–O bond order for the McClure structure are correlated with measured Fe–O and O–O bond lengths for oxymyoglobin. Back-bonding from O ₂ ^? to Fe^III of the Weiss structure gives a restricted form of the McClure structure. The McClure and Weiss increased-valence structures are used to provide VB formulations of mechanisms for the oxyhemoglobin + NO reaction. The products of these two formulations are Hb⁺ and NO₃ ^? (where Hb is hemoglobin) and Hb⁺ and OONO^?, respectively. Because Hb⁺ and NO₃ ^? are the observed products, they provide an experimental procedure for distinguishing the McClure and Weiss models. It is also shown that the same type of agreement between McClure-type theory and experiment occurs for oxycoboglobin + NO, cytochrome P450 monooxygenases, and related hydrogen atom transfer reactions. In the appendices, the results of density functional theory and multireference molecular orbital calculations for oxyhemes are related to one formulation of the increased-valence wavefunction for the McClure model, and theory is presented for the calculation of approximate weights for the Lewis structures that are components of the McClure increased-valence structure.     

Methemoglobin formation in mutant hemoglobin α chains: electron transfer parameters and rates

Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.     

Synthesis and structures of dimeric iron(III)-Oxo and -imido complexes containing intramolecular hydrogen bonds

Hydrogen bonding networks proximal to metal centers are emerging as a viable means for controlling secondary coordination spheres. This has led to the regulation of reactivity and isolation of complexes with new structural motifs. We have used the tridenate ligand bis[(N′-tert-butylureido)-N-ethyl]-N-methylaminato ([H₂1]²⁻) that contains two hydrogen bond donors to examine the oxidation of the Fe^II-acetate complex, [Fe^IIH₂1(η²-OAc)]⁻ with dioxygen, amine N-oxides, and xylyl azide. A complex with Fe^III-O-Fe^III core results from the oxidation with dioxygen and amine N-oxides, in which the oxo ligand is involved in hydrogen bonding to the [H₂1]²⁻ ligand. A distinctly different hydrogen bonding network was found in Fe^III dimer isolated from the reaction with the xylyl azide: a rare Fe^III-N(R)-Fe^III core was observed that does not have hydrogen bonds to the bridging nitrogen atom. The intramolecular H-bond networks within these dimers appear to adjust to the presence of the bridging species and rearrange to its size and electron density.     

Structures and properties of octaethylporphinato(phenolate)iron(III) complexes with NH?O hydrogen bonds: modulation of Fe-O bond character by the hydrogen bond

Iron(III) porphinate complexes of phenolate that have NH?O hydrogen bonds on the coordinating oxygen, [Fe^III(OEP){O-2,6-(RCONH)₂C₆H₃}] (R = CF₃ (1), CH₃ (3)) and [Fe^III(OEP)(O-2-RCONHC₆H₄)] (R = CF₃ (2), CH₃ (4)) (OEP = 2,3,7,8,12,13,17,18-octaethyl-21H, 23H-porphinato), were synthesized and characterized as models of heme catalase. The presence of NH?O hydrogen bonds was established by their crystal structures and IR shifts of the amide NH band. The crystal structure of 1 shows an extremely elongated Fe-O bond, 1.926(3) Å, compared to 1.887(2) Å in 2 or 1.848(4) Å in [Fe^III(OEP)(OPh)]. The NH?O hydrogen bond decreases an electron donation from oxygen to iron, resulting in a long Fe-O bond and a positive redox potential.     

Nitric Oxide Binds to the Proximal Heme Coordination Site of the Ferrocytochrome c/Cardiolipin Complex: FORMATION MECHANISM AND DYNAMICS*

Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 × 10⁷ m⁻¹ s⁻¹). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c′ and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin.     

Iron-induced oxidation of (all-E)-β-carotene under model gastric conditions: kinetics,products, and mechanism

The stability of (all-E)-β-carotene toward dietary iron was studied in a mildly acidic (pH 4) micellar solution as a simple model of the postprandial gastric conditions. The oxidation was initiated by free iron (Fe^II, Fe^III) or by heme iron (metmyoglobin, MbFe^III). Fe^II and metmyoglobin were much more efficient than Fe^III at initiating β-carotene oxidation. Whatever the initiator, hydrogen peroxide did not accumulate. Moreover, β-carotene markedly inhibited the conversion of Fe^II into Fe^III. β-Carotene oxidation induced by Fe^II or MbFe^III was maximal with 5–10 eq Fe^II or 0.05–0.1 eq MbFe^III and was inhibited at higher iron concentrations, especially with Fe^II. UPLC/DAD/MS and GC/MS analyses revealed a complex distribution of β-carotene-derived products including Z-isomers, epoxides, and cleavage products of various chain lengths. Finally, the mechanism of iron-induced β-carotene oxidation is discussed. Altogether, our results suggest that dietary iron, especially free (loosely bound) Fe^II and heme iron, may efficiently induce β-carotene autoxidation within the upper digestive tract, thereby limiting its supply to tissues (bioavailability) and consequently its biological activity.     

Capturing the spin state diversity of iron(III)-aryl porphyrins: OLYP is better than TPSSh

DFT calculations with a variety of exchange-correlation functionals, including PW91, OLYP, TPSSh, B3LYP and B3LYP*, have been carried out on the low-energy spin states of chloroiron(III) porphyrin and four aryliron(III) porphyrins, viz. Fe^III(P)Ph (S = 1/2), Fe^III(P)C₆F₅ (S = 5/2), Fe^III(P)(3,4,5-C₆F₃H₂) (S = 1/2), Fe^III(P)(2,4,6-C₆F₃H₂) (S = 5/2), where the expected spin states have been indicated within parentheses. Qualitatively, OLYP reproduces all the expected ground spin states. B3LYP appears to have some difficulty yielding the observed sextet ground states. B3LYP*, TPSSh and PW91 all fail to reproduce the sextet ground states, the latter two by rather large margins of energy. As far as this study is concerned, the overall performance of the functionals appears to be OLYP/OPBE > B3LYP > B3LYP* >> TPSSh > PW91/BLYP/BP86/TPSS.     

Methemoglobin formation in mutant hemoglobin α chains: electron transfer parameters and rates

Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.     

Nitrite reduction by Co(II) and Mn(II) substituted myoglobins: towards understanding necessary components of Mb nitrite reductase activity

Nitrite reduction to nitric oxide by heme proteins is drawing increasing attention as a protective mechanism to hypoxic injury in mammalian physiology. Here we probe the nitrite reductase (NiR) activities of manganese(II)- and cobalt(II)-substituted myoglobins, and compare with data obtained previously for the iron(II) analog wt Mb^II. Both Mn^IIMb and Co^IIMb displayed NiR activity, and it was shown that the kinetics are first order each in [protein], [nitrite], and [H⁺], as previously determined for the Fe^II analog wt Mb^II. The second order rate constants (k₂) at pH 7.4 and T = 25 °C, were 0.0066 and 0.015 M^− 1 s^− 1 for Co^IIMb and Mn^IIMb, respectively, both orders of magnitude slower than the k₂ (6 M^− 1 s^− 1) for wt Mb^II. The final reaction products for Mn^IIMb consisted of a mixture of the nitrosyl Mn^IIMb(NO) and Mn^IIIMb, similar to the products from the analogous NiR reaction by wt Mb. In contrast, the products of NiR by Co^IIMb were found to be the nitrito complex Co^IIIMb(ONO⁻) plus roughly an equivalent of free NO. The differences can be attributed in part to the stronger coordination of inorganic nitrite to Co^IIIMb as reflected in the respective M^IIIMb(ONO⁻) formation constants K_nitrite: 2100 M^− 1 (Co^III) and <~0.4 M^− 1 (Mn^III). We also report the formation constants (3.7 and 30 M^− 1, respectively) for the nitrite complexes of the mutant metmyoglobins H64V Mb^III(NO₂⁻) and H64V/V67R Mb^III(ONO⁻) and a K_nitrite revised value (120 M^− 1) for the nitrite complex of wt metMb. The respective K_nitrite values for the three ferric proteins emphasize the importance of a H-bonding residue, such as His64 in the Mb^III distal pocket or the Arg67 in H64V/V67R Mb^III, in stabilizing nitrite coordination. Notably, the NiR activities of the corresponding ferrous Mbs follow a similar sequence suggesting that nitrite binding to these centers are analogously affected by the H-bonding residues.     

The interaction of native calf thymus DNA with Fe(III)-dipyrido[3,2-a:2',3'-c]phenazine

The mono and bis dipyrido[3,2-a:2′,3′-c]phenazine (dppz) adducts of iron(III) chloride, i.e. [Fe(dppz)]Cl₃ and [Fe(dppz)₂]Cl₃, have been synthesized and characterized. The interaction of the Fe^IIIdppz hydrolyzed aquo complex with native calf thymus DNA has been monitored as a function of the metal complex-DNA molar ratio, by variable temperature UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results obtained in solution at various ionic strength values give support for a tight intercalative binding of the Fe^IIIdppz cation with DNA. In particular, the appearance of induced CD bands, caused by the addition of Fe^IIIdppz, indicate the existence of a rigid metal complex-DNA-binding leading to dominating chiral organization of Fe^IIIdppz species within the DNA double helix. The trend of selected CD bands with the molar concentration of Fe^IIIdppz emphasizes that the presence of high amounts of metal complex induces also the formation of DNA-Fe^IIIdppz supramolecular aggregates in solution. The analysis of fluorescence measurements allowed us to calculate a value of the intercalative binding constant comparable to that obtained by UV spectrophotometric titration. Finally, the temperature dependence of the absorbance at 258 nm shows that the metal complex strongly increases the DNA melting temperature already at metal complex-DNA molar ratio equal to 0.25 suggesting that metal complex intercalation effectively hinders DNA denaturation. Overall, the results of the present study point out that the Fe^IIIdppz aquo complex has DNA-binding properties analogous to those previously reported for the tris-chelate Fe^II(phen)₂dppz complex (phen = 1,10-phenantroline).