Iron hemiporphycene as a functional prosthetic group for myoglobin

The iron complex of hemiporphycene, a molecular hybrid of porphyrin with porphycene, was incorporated into the apomyoglobin pocket to examine ligand binding ability of the iron atom in the novel porphyrinoid. Apomyoglobin was successfully coupled with a stoichiometric amount of ferric hemiporphycene to afford the reconstituted myoglobin equipped with the iron coordination structure of native protein. Cyanide, imidazole, and fluoride coordinated to the ferric protein with affinities comparable with those for native myoglobin. The ferrous myoglobin was functionally active to bind O(2) and CO reversibly at pH 7.4 and 20 degrees C. The O(2) affinity is 12-fold higher than that of native myoglobin while the CO affinity is slightly lower, suggesting decreased discrimination between O(2) and CO in the heme pocket. The functional anomaly was interpreted to reflect increased sigma-bonding character in the Fe(II)-O(2) bond. In contrast with 6-coordinate native NO protein, the NO myoglobin containing ferrous hemiporphycene is in a mixed 5- and 6-coordinate state. This observation suggests that the in-plane configuration of the iron atom in hemiporphycene is destabilized by NO. Influence of the core deformation was also detected with both the infrared absorption for the ferrous CO derivative and electron paramagnetic resonance for ferric imidazole complex. Anomalies in the ferric and ferrous derivatives were ascribed to the modified iron-N(pyrrole) interactions in the asymmetric metallo core of hemiporphycene.     

Reactions of nitrogen oxides with the five-coordinate Fe(III)(porphyrin) nitrito intermediate Fe(Por)(ONO) in sublimed solids

Detailed experimental studies are described for reactions of several nitrogen oxides with iron porphyrin models for heme/NxOy systems. It is shown by FTIR and optical spectroscopy and by isotope labeling experiments that reaction of small increments of NO2 with sublimed thin layers of the iron(II) complex Fe(Por) (Por = meso-tetraphenylporphyrinato dianion, TPP, or meso-tetra-p-tolylporphyrinato dianion, TTP) leads to formation of the 5-coordinate nitrito complexes Fe(Por)(eta1-ONO) (1), which are fairly stable but very slowly decompose under vacuum giving mostly the corresponding nitrosyl complexes Fe(Por)(NO). Further reaction of 1 with new NO2 increments leads to formation of the nitrato complex Fe(Por)(eta2-O2NO) (2). The interaction of NO with 1 at low temperature involves ligand addition to give the nitrito-nitrosyl complexes Fe(Por)(eta1-ONO)(NO) (3); however, these isomerize to the nitro-nitrosyl analogs Fe(Por)(eta1-NO2)(NO) (4) upon warming. Experiments with labeled nitrogen oxides argue for an intramolecular isomerization ("flipping") mechanism rather than one involving dissociation and rebinding of NO2. The Fe(III) centers in the 6-coordinate species 3 and 4 are low spin in contrast to 1, which appears to be high-spin, although DFT computations of the porphinato models Fe(P)(nitrite) suggest that the doublet nitro species and the quartet and sextet nitrito complexes are all relatively close in energy. The nitro-nitrosyl complex 4 is stable under an NO atmosphere but decomposes under intense pumping to give a mixture of the ferrous nitrosyl complex Fe(Por)(NO) and the ferric nitrito complex Fe(Por)(eta1-ONO) indicating the competitive dissociation of NO and NO2. Hence, loss of NO from 4 is accompanied with nitro --> nitrito isomerization consistent with 1 being the more stable of the 5-coordinate NO2 complexes of iron porphyrins.     

Electronic structure of six-coordinate iron(III)-porphyrin NO adducts: the elusive iron(III)-NO(radical) state and its influence on the properties of these complexes

This paper investigates the interaction between five-coordinate ferric hemes with bound axial imidazole ligands and nitric oxide (NO). The corresponding model complex, [Fe(TPP)(MI)(NO)](BF4) (MI = 1-methylimidazole), is studied using vibrational spectroscopy coupled to normal coordinate analysis and density functional theory (DFT) calculations. In particular, nuclear resonance vibrational spectroscopy is used to identify the Fe-N(O) stretching vibration. The results reveal the usual Fe(II)-NO(+) ground state for this complex, which is characterized by strong Fe-NO and N-O bonds, with Fe-NO and N-O force constants of 3.92 and 15.18 mdyn/A, respectively. This is related to two strong pi back-bonds between Fe(II) and NO(+). The alternative ground state, low-spin Fe(III)-NO(radical) (S = 0), is then investigated. DFT calculations show that this state exists as a stable minimum at a surprisingly low energy of only approximately 1-3 kcal/mol above the Fe(II)-NO(+) ground state. In addition, the Fe(II)-NO(+) potential energy surface (PES) crosses the low-spin Fe(III)-NO(radical) energy surface at a very small elongation (only 0.05-0.1 A) of the Fe-NO bond from the equilibrium distance. This implies that ferric heme nitrosyls with the latter ground state might exist, particularly with axial thiolate (cysteinate) coordination as observed in P450-type enzymes. Importantly, the low-spin Fe(III)-NO(radical) state has very different properties than the Fe(II)-NO(+) state. Specifically, the Fe-NO and N-O bonds are distinctively weaker, showing Fe-NO and N-O force constants of only 2.26 and 13.72 mdyn/A, respectively. The PES calculations further reveal that the thermodynamic weakness of the Fe-NO bond in ferric heme nitrosyls is an intrinsic feature that relates to the properties of the high-spin Fe(III)-NO(radical) (S = 2) state that appears at low energy and is dissociative with respect to the Fe-NO bond. Altogether, release of NO from a six-coordinate ferric heme nitrosyl requires the system to pass through at least three different electronic states, a process that is remarkably complex and also unprecedented for transition-metal nitrosyls. These findings have implications not only for heme nitrosyls but also for group-8 transition-metal(III) nitrosyls in general.     

A quantum chemical survey of metalloporphyrin-nitrosyl linkage isomers: insights into the observation of multiple FeNO conformations in a recent crystallographic determination of nitrophorin 4

Using density functional theory-based geometry optimizations, we have searched for eta(1)-NO, eta(1)-ON (isonitrosyl), and eta(2)-NO (side-on bound NO) linkage isomers of a number of metalloporphyrin-NO complexes, M(Por)(NO)(L), where Por = porphinato dianion, M = Mn(II), Fe(II), Fe(III), Ru(II), Ru(III), Co(II), and Rh(II), and L = no ligand, SMe, Ph, and imidazole. The eta(1)-NO isomer had the lowest energy in all cases, and the isonitrosyl isomer was also located as a higher energy potential energy minimum in a number of cases. The eta(2)-NO isomer was only located as a minimum for Mn(II) (L = no ligand), Fe(III) (L = no ligand), and Ru(III) (L = Ph, imidazole, pyrdine), suggesting that an [MNO](6) electron count is important for stabilization of the eta(2) mode of ligation. However, in the presence of axial ligands L, the side-on isomers of [FeNO](6) complexes were not stable and opened up to an unusual geometry where the FeN(O) and NO vectors were tilted in opposite directions relative to the heme normal. Exactly such a geometry, as well as a "normal" upright geometry, has been observed in a recent crystallographic determination of nitrophorin 4 (Nature Struct. Biol. 2000, 7, 551), a salivary protein from the blood-sucking insect Rhodnius prolixus. Together, the calculated and experimental result illustrate the extreme softness of the FeNO potential energy surface toward various forms of tilting and bending deformations.     

Alkylamine-ligated H93G myoglobin cavity mutant: a model system for endogenous lysine and terminal amine ligation in heme proteins such as nitrite reductase and cytochrome f

His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, providing a remarkably versatile template for the preparation of model heme complexes. The investigation of model heme adducts is an important way to probe the relationship between coordination structure and catalytic function in heme enzymes. In this study, we have successfully generated and spectroscopically characterized the H93G Mb cavity mutant ligated with less common alkylamine ligands (models for Lys or the amine group of N-terminal amino acids) in numerous heme iron states. All complexes have been characterized by electronic absorption and magnetic circular dichroism spectroscopy in comparison with data for parallel imidazole-ligated H93G heme iron moieties. This is the first systematic spectral study of models for alkylamine- or terminal amine-ligated heme centers in proteins. High-spin mono- and low-spin bis-amine-ligated ferrous and ferric H93G Mb adducts have been prepared together with mixed-ligand ferric heme complexes with alkylamine trans to nitrite or imidazole as heme coordination models for cytochrome c nitrite reductase or cytochrome f, respectively. Six-coordinate ferrous H93G Mb derivatives with CO, NO, and O(2) trans to the alkylamine have also been successfully formed, the latter for the first time. Finally, a novel high-valent ferryl species has been generated. The data in this study represent the first thorough investigation of the spectroscopic properties of alkylamine-ligated heme iron systems as models for naturally occurring heme proteins ligated by Lys or terminal amines.     

Studies of iron(II) and iron(III) complexes with fac-N2O, cis-N2O2 and N2O3 donor ligands: models for the 2-His 1-carboxylate motif of non-heme iron monooxygenases

Enzymes in the oxygen-activating class of mononuclear non-heme iron oxygenases (MNOs) contain a highly conserved iron center facially ligated by two histidine nitrogen atoms and one carboxylate oxygen atom that leave one face of the metal center (three binding sites) open for coordination to cofactor, substrate, and/or dioxygen. A comparative family of [Fe(II/III)(N(2)O(n))(L)(4-n))](±x), n = 1-3, L = solvent or Cl(-), model complexes, based on a ligand series that supports a facially ligated N,N,O core that is then modified to contain either one or two additional carboxylate chelate arms, has been structurally and spectroscopically characterized. EPR studies demonstrate that the high-spin d(5) Fe(III)g = 4.3 signal becomes more symmetrical as the number of carboxylate ligands decreases across the series Fe(N(2)O(3)), Fe(N(2)O(2)), and Fe(N(2)O(1)), reflecting an increase in the E/D strain of these complexes as the number of exchangeable/solvent coordination sites increases, paralleling the enhanced distribution of electronic structures that contribute to the spectral line shape. The observed systematic variations in the Fe(II)-Fe(III) oxidation-reduction potentials illustrate the fundamental influence of differential carboxylate ligation. The trend towards lower reduction potential for the iron center across the [Fe(III)(N(2)O(1))Cl(3)](-), [Fe(III)(N(2)O(2))Cl(2)](-) and [Fe(III)(N(2)O(3))Cl](-) series is consistent with replacement of the chloride anions with the more strongly donating anionic O-donor carboxylate ligands that are expected to stabilize the oxidized ferric state. This electrochemical trend parallels the observed dioxygen sensitivity of the three ferrous complexes (Fe(II)(N(2)O(1)) < Fe(II)(N(2)O(2)) < Fe(II)(N(2)O(3))), which form μ-oxo bridged ferric species upon exposure to air or oxygen atom donor (OAD) molecules. The observed oxygen sensitivity is particularly interesting and discussed in the context of α-ketoglutarate-dependent MNO enzyme mechanisms.     

Coordination versatility of tridentate pyridyl aroylhydrazones towards iron: tracking down the elusive aroylhydrazono-based ferric spin-crossover molecular materials

The two potentially tridentate and monoprotic Schiff bases acetylpyridine benzoylhydrazone (HL(1)) and acetylpyridine 4-tert-butylbenzoylhydrazone (HL(2)) demonstrate remarkable coordination versatility towards iron on account of their propensity to undergo tautomeric transformations as imposed by the metal centre. Each of the pyridyl aroylhydrazone ligands complexes with the ferrous or ferric ion under strictly controlled reaction conditions to afford three six-coordinate mononuclear compounds [Fe(II)(HL)(2)](ClO(4))(2), [Fe(II)L(2)] and [Fe(III)L(2)]ClO(4) (HL = HL(1) or HL(2)) displaying distinct colours congruent with their intense CT visible absorptions. The synthetic manoeuvres rely crucially on the stoichiometry of the reactants, the basicities of the reaction mixtures and the choice of solvent. Electrochemically, each of these iron compounds exhibits a reversible metal-centred redox process. By all appearances, [Fe(III)(L(1))(2)]ClO(4) is one of only two examples of a crystallographically elucidated iron(III) bis-chelate compound of a pyridyl aroylhydrazone. Several pertinent physical measurements have established that each of the Schiff bases stabilises multiple spin states of iron; the enolate form of these ligands exhibits greater field strength than does the corresponding neutral keto tautomer. To the best of our knowledge, [Fe(III)(L(1))(2)]ClO(4) and [Fe(III)(L(2))(2)]ClO(4) are the first examples of ferric spin crossovers of aroylhydrazones. Whereas in the former the spin crossover (SCO) is an intricate gradual process, in the latter the (6)A(1)?(2)T(2) transition curve is sigmoidal with T(?)～280 K and the SCO is virtually complete. As regards [Fe(III)(L(1))(2)]ClO(4), M?ssbauer and EPR spectroscopic techniques have revealed remarkable dependence of the spin transition on sample type and extent of solvation. In frozen MeOH solution at liquid nitrogen temperature, both iron(III) compounds exist wholly in the doublet ground state.     

Synthesis, structure, and properties of an Fe(II) carbonyl [(PaPy3)Fe(CO)](ClO4): insight into the reactivity of Fe(II)-CO and Fe(II)-NO moieties in non-heme iron chelates of N-donor ligands

An Fe(II) carbonyl complex [(PaPy3)Fe(CO)](ClO4) (1) of the pentadentate ligand N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide (PaPy3H, H is the dissociable amide proton) has been synthesized and structurally characterized. This Fe(II) carbonyl exhibits its nu(CO) at 1972 cm(-1), and its 1H NMR spectrum in degassed CD3CN confirms its S = 0 ground state. The bound CO in 1 is not photolabile. Reaction of 1 with an equimolar amount of NO results in the formation of the {Fe-NO}7 nitrosyl [(PaPy3)Fe(NO)](ClO4) (2), while excess NO affords the iron(III) nitro complex [(PaPy3)Fe(NO2)](ClO4) (5). In the presence of [Fe(Cp)2]+ and excess NO, 1 forms the {Fe-NO}6 nitrosyl [(PaPy3)Fe(NO)](ClO4)2 (3). Complex 1 also reacts with dioxygen to afford the iron(III) mu-oxo species [{(PaPy3)Fe}2O](ClO4)2 (4). Comparison of the metric and spectral parameters of 1 with those of the previously reported {Fe-NO}6,7 nitrosyls 3 and 2 provides insight into the electronic distributions in the Fe(II)-CO, Fe(II)-NO, and Fe(II)-NO+ bonds in the isostructural series of complexes 1-3 derived from a non-heme polypyridine ligand with one carboxamide group.     

X-ray absorption spectroscopy of hemes and hemeproteins in solution: multiple scattering analysis

A full quantitative analysis of Fe K-edge X-ray absorption spectra has been performed for hemes in two porphynato complexes, that is, iron(III) tetraphenylporphyrin chloride (Fe(III)TPPCl) and iron(III) tetraphenylporphyrin bis(imidazole) (Fe(III)TPP(Imid)2), in two protein complexes whose X-ray structure is known at atomic resolution (1.0 A), that is, ferrous deoxy-myoglobin (Fe(II)Mb) and ferric aquo-myoglobin (Fe(III)MbH2O), and in ferric cyano-myoglobin (Fe(III)MbCN), whose X-ray structure is known at lower resolution (1.4 A). The analysis has been performed via the multiple scattering approach, starting from a muffin tin approximation of the molecular potential. The Fe-heme structure has been obtained by analyzing independently the Extended X-ray Absorption Fine Structure (EXAFS) region and the X-ray Absorption Near Edge Structure (XANES) region. The EXAFS structural results are in full agreement with the crystallographic values of the models, with an accuracy of +/- 0.02 A for Fe-ligand distances, and +/-6 degrees for angular parameters. All the XANES features above the theoretical zero energy (in the lower rising edge) are well accounted for by single-channel calculations, for both Fe(II) and Fe(III) hemes, and the Fe-N p distance is determined with the same accuracy as EXAFS. XANES evaluations of Fe-5th and Fe-6th ligand distances are determined with 0.04-0.07 A accuracy; a small discrepancy with EXAFS (0.01 to 0.05 A beyond the statistical error), is found for protein compounds. Concerns from statistical correlation among parameters and multiple minima in the parameter space are discussed. As expected, the XANES accuracy is slightly lower than what was found for polarized XANES on Fe(III)MbCN single crystal (0.03-0.04 A), and states the actual state-of-the-art of XANES analysis when used to extract heme-normal parameters in a solution spectrum dominated by heme-plane scattering.     

Enrichment of phosphopeptides using bare magnetic particles

Magnetic iron(II, III) oxide (magnetite, Fe(3)O(4)) nanoparticles were used to selectively enrich phosphopeptides from tryptic digests of bovine beta-casein and from tryptic digest mixtures containing bovine beta-casein, cytochrome c, bovine serum albumin, and horse heart myoglobin. The magnetic property of the particles permits an easy and speedy enrichment process. No enrichment of phosphopeptides was observed from ferric magnetic iron(III) oxide (Fe(2)O(3)) nanoparticles. These data collectively demonstrate that the enrichment of phosphopeptides using magnetic iron(II, III) oxide nanoparticles is a practical method for the selective analysis of phosphopeptides and could be helpful in isolating and analyzing phosphorylated peptides from complex biological samples.     

Tuning the electronic structure of octahedral iron complexes [FeL(X)] (L = 1-alkyl-4,7-bis(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane,X = Cl,CH(3)O,CN, NO). The S = 1/2 <==>3/2 Spin equilibrium of [FeL(Pr)(NO)

Two new pentadentate, pendent arm macrocyclic ligands of the type 1-alkyl-4,7-bis(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane where alkyl represents an isopropyl, (L(Pr))(2-), or an ethyl group, (L(Et))(2-), have been synthesized. It is shown that they bind strongly to ferric ions generating six-coordinate species of the type [Fe(L(alk))X]. The ground state of these complexes is governed by the nature of the sixth ligand, X: [Fe(III)(L(Et))Cl] (2) possesses an S = 5/2 ground state as do [Fe(III)(L(Et))(OCH(3))] (3) and [Fe(III)(L(Pr))(OCH(3))] (4). In contrast, the cyano complexes [Fe(III)(L(Et))(CN)] (5) and [Fe(III)(L(Pr))(CN)] (6) are low spin ferric species (S = 1/2). The octahedral [FeNO](7) nitrosyl complex [Fe(L(Pr))(NO)] (7) displays spin equilibrium behavior S = 1/2<==>S = (3)/(2) in the solid state. Complexes [Zn(L(Pr))] (1), 4.CH(3)OH, 5.0.5toluene.CH(2)Cl(2), and 7.2.5CH(2)Cl(2) have been structurally characterized by low-temperature (100 K) X-ray crystallography. All iron complexes have been carefully studied by zero- and applied-field M?ssbauer spectroscopy. In addition, Sellmann's complexes [Fe(pyS(4))(NO)](0/1+) and [Fe(pyS(4))X] (X = PR(3), CO, SR(2)) have been studied by EPR and M?ssbauer spectroscopies and DFT calculations (pyS(4) = 2,6-bis(2-mercaptophenylthiomethyl)pyridine(2-)). It is concluded that the electronic structure of 7 with an S = 1/2 ground state is low spin ferrous (S(Fe) = 0) with a coordinated neutral NO radical (Fe(II)-NO) whereas the S = 3/2 state corresponds to a high spin ferric (S(Fe) = 5/2) antiferromagnetically coupled to an NO(-) anion (S = 1). The S = 1/2<==>S = 3/2 equilibrium is then that of valence tautomers rather than that of a simple high spin<==>low spin crossover.     

Ligand binding properties of myoglobin reconstituted with iron porphycene: unusual O2 binding selectivity against CO binding

Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild-type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O2 affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O2 dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O2 affinity of the reconstituted myoglobin exceeds its CO affinity (M' = K(CO)/K(O2) < 1). The ligand binding studies on H64A mutants support the fact that the slow O2 dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O2 sigma-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O2 affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein.     

Iron nitrosyls of a pentadentate ligand containing a single carboxamide group: syntheses, structures, electronic properties, and photolability of NO

Three iron complexes of a pentadentate ligand N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide (PaPy(3)H, H is the dissociable amide proton) have been synthesized. All three species, namely, two nitrosyls [(PaPy(3))Fe(NO)](ClO(4))(2) (2) and [(PaPy(3))Fe(NO)](ClO(4)) (3) and one nitro complex [(PaPy(3))Fe(NO(2))](ClO(4)) (4), have been structurally characterized. These complexes provide the opportunity to compare the structural and spectral properties of a set of isostructural [Fe-NO](6,7) complexes (2 and 3, respectively) and an analogous genuine Fe(III) complex with an "innocent" sixth ligand ([(PaPy(3))Fe(NO(2))](ClO(4)), 4). The most striking difference in the structural features of 2 and 3 is the Fe-N-O angle (Fe-N-O = 173.1(2) degrees in the case of 2 and 141.29(15) degrees in the case of 3). The clean (1)H NMR spectrum of 2 in CD(3)CN reveals its S = 0 ground state and confirms its [Fe-NO](6) configuration. The binding of NO at the non-heme iron center in 2 is completely reversible and the bound NO is photolabile. M?ssbauer data, electron paramagnetic resonance signal at g approximately 2.00, and variable temperature magnetic susceptibility measurements indicate the S = (1)/(2) spin state of the [Fe-NO](7) complex 3. Analysis of the spectroscopic data suggests Fe(II)-NO(+) and Fe(II)-NO(*) formulations for 2 and 3, respectively. The bound NO in 3 does not show any photolability. However, in MeCN solution, it reacts rapidly with dioxygen to afford the nitro complex 4, which has also been synthesized independently from [(PaPy(3))Fe(MeCN)](2+) and NO(2)(-). Nucleophilic attack of hydroxide ion to the N atom of the NO ligand in 2 in MeCN in the dark gives rise to 4 in high yield.     

Enterobactin protonation and iron release: structural characterization of the salicylate coordination shift in ferric enterobactin

The siderophore enterobactin (Ent) is produced by many species of enteric bacteria to mediate iron uptake. This iron scavenger can be reincorporated by the bacteria as the ferric complex [Fe(III)(Ent)](3)(-) and is subsequently hydrolyzed by an esterase to facilitate intracellular iron release. Recent literature reports on altered protein recognition and binding of modified enterobactin increase the significance of understanding the structural features and solution chemistry of ferric enterobactin. The structure of the neutral protonated ferric enterobactin complex [Fe(III)(H(3)Ent)](0) has been the source of some controversy and confusion in the literature. To demonstrate the proposed change of coordination from the tris-catecholate [Fe(III)(Ent)](3)(-) to the tris-salicylate [Fe(III)(H(3)Ent)](0) upon protonation, the coordination chemistry of two new model compounds N,N',N'-tris[2-(hydroxybenzoyl)carbonyl]cyclotriseryl trilactone (SERSAM) and N,N',N'-tris[2-hydroxy,3-methoxy(benzoyl)carbonyl]cyclotriseryl trilactone (SER(3M)SAM) was examined in solution and solid state. Both SERSAM and SER(3M)SAM form tris-salicylate ferric complexes with spectroscopic and solution thermodynamic properties (with log beta(110)() values of 39 and 38 respectively) similar to those of [Fe(III)(H(3)Ent)](0). The fits of EXAFS spectra of the model ferric complexes and the two forms of ferric enterobactin provided bond distances and disorder factors in the metal coordination sphere for both coordination modes. The protonated [Fe(III)(H(3)Ent)](0) complex (d(Fe)(-)(O) = 1.98 A, sigma(2)(stat)(O) = 0.00351(10) A(2)) exhibits a shorter average Fe-O bond length but a much higher static Debye-Waller factor for the first oxygen shell than the catecholate [Fe(III)(Ent)](3)(-) complex (d(Fe)(-)(O) = 2.00 A, sigma(2)(stat)(O) = 0.00067(14) A(2)). (1)H NMR spectroscopy was used to monitor the amide bond rotation between the catecholate and salicylate geometries using the gallic complexes of enterobactin: [Ga(III)(Ent)](3)(-) and [Ga(III)(H(3)Ent)](0). The ferric salicylate complexes display quasi-reversible reduction potentials from -89 to -551 mV (relative to the normal hydrogen electrode NHE) which supports the feasibility of a low pH iron release mechanism facilitated by biological reductants.     

Distinct reaction pathways followed upon reduction of oxy-heme oxygenase and oxy-myoglobin as characterized by Mössbauer spectroscopy

Activation of O(2) by heme-containing monooxygenases generally commences with the common initial steps of reduction to the ferrous heme and binding of O(2) followed by a one-electron reduction of the O(2)-bound heme. Subsequent steps that generate reactive oxygen intermediates diverge and reflect the effects of protein control on the reaction pathway. In this study, M?ssbauer and EPR spectroscopies were used to characterize the electronic states and reaction pathways of reactive oxygen intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of oxy-heme oxygenase (HO) and oxy-myoglobin (Mb). The results confirm that one-electron reduction of (Fe(II)-O(2))HO is accompanied by protonation of the bound O(2) to generate a low-spin (Fe(III)-O(2)H(-))HO that undergoes self-hydroxylation to form the alpha-meso-hydroxyhemin-HO product. In contrast, one-electron reduction of (Fe(II)-O(2))Mb yields a low-spin (Fe(III)-O(2)(2-))Mb. Protonation of this intermediate generates (Fe(III)-O(2)H(-))Mb, which then decays to a ferryl complex, (Fe(IV)=O(2-))Mb, that exhibits magnetic properties characteristic of the compound II species generated in the reactions of peroxide with heme peroxidases and with Mb. Generation of reactive high-valent states with ferryl species via hydroperoxo intermediates is believed to be the key oxygen-activation steps involved in the catalytic cycles of P450-type monooxygenases. The M?ssbauer data presented here provide direct spectroscopic evidence supporting the idea that ferric-hydroperoxo hemes are indeed the precursors of the reactive ferryl intermediates. The fact that a ferryl intermediate does not accumulate in HO underscores the determining role played by protein structure in controlling the reactivity of reaction intermediates.     

Geometric and electronic structure of the heme-peroxo-copper complex [(F8TPP)FeIII-(O22-)-CuII(TMPA)](ClO4)

The geometric and electronic structure of the untethered heme-peroxo-copper model complex [(F(8)TPP)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](ClO(4)) (1) has been investigated using Cu and Fe K-edge EXAFS spectroscopy and density functional theory calculations in order to describe its geometric and electronic structure. The Fe and Cu K-edge EXAFS data were fit with a Cu...Fe distance of approximately 3.72 A. Spin-unrestricted DFT calculations for the S(T) = 2 spin state were performed on [(P)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](+) as a model of 1. The peroxo unit is bound end-on to the copper, and side-on to the high-spin iron, for an overall mu-eta(1):eta(2) coordination mode. The calculated Cu...Fe distance is approximately 0.3 A longer than that observed experimentally. Reoptimization of [(P)Fe(III)-(O(2)(2)(-))-Cu(II)(TMPA)](+) with a 3.7 A Cu...Fe constrained distance results in a similar energy and structure that retains the overall mu-eta(1):eta(2)-peroxo coordination mode. The primary bonding interaction between the copper and the peroxide involves electron donation into the half-occupied Cu d(z)2 orbital from the peroxide pi(sigma) orbital. In the case of the Fe(III)-peroxide eta(2) bond, the two major components arise from the donor interactions of the peroxide pi*(sigma) and pi*(v) orbitals with the Fe d(xz) and d(xy) orbitals, which give rise to sigma and delta bonds, respectively. The pi*(sigma) interaction with both the half-occupied d(z)2 orbital on the copper (eta(1)) and the d(xz) orbital on the iron (eta(2)), provides an effective superexchange pathway for strong antiferromagnetic coupling between the metal centers.     

Is histidine dissociation a critical component of the NO/H-NOX signaling mechanism? Insights from X-ray absorption spectroscopy

The H-NOX (Heme-Nitric oxide/OXygen binding) family of diatomic gas sensing hemoproteins has attracted great interest. Soluble guanylate cyclase (sGC), the well-characterized eukaryotic nitric oxide (NO) sensor is an H-NOX family member. When NO binds sGC at the ferrous histidine-ligated protoporphyrin-IX, the proximal histidine ligand dissociates, resulting in a 5-coordinate (5c) complex; formation of this 5c complex is viewed as necessary for activation of sGC. Characterization of other H-NOX family members has revealed that while most also bind NO in a 5c complex, some bind NO in a 6-coordinate (6c) complex or as a 5c/6c mixture. To gain insight into the heme pocket structural differences between 5c and 6c Fe(ii)-NO H-NOX complexes, we investigated the extended X-ray absorption fine structure (EXAFS) of the Fe(II)-unligated and Fe(II)-NO complexes of H-NOX domains from three species, Thermoanaerobacter tengcongensis, Shewanella woodyi, and Pseudoalteromonas atlantica. Although the Fe(II)-NO complex of TtH-NOX is formally 6c, we found the Fe-N(His) bond is substantially lengthened. Furthermore, although NO binds to SwH-NOX and PaH-NOX as a 5c complex, consistent with histidine dissociation, the EXAFS data do not exclude a very weakly associated histidine. Regardless of coordination number, upon NO-binding, the Fe-N(porphyrin) bond lengths in all three H-NOXs contract by ~0.07 ?. This study reveals that the overall heme structure of 5c and 6c Fe(II)-NO H-NOX complexes are substantially similar, suggesting that formal histidine dissociation may not be required to trigger NO/H-NOX signal transduction. The study has refined our understanding of the molecular mechanisms underlying NO/H-NOX signaling.     

pH controls the rate and mechanism of nitrosylation of water-soluble FeIII porphyrin complexes

In-depth kinetic and mechanistic studies on the reversible binding of NO to water-soluble iron(III) porphyrins as a function of pH revealed unexpected reaction kinetics for monohydroxo-ligated (P)Fe(III)(OH) species formed by deprotonation of coordinated water in diaqua-ligated (P)Fe(III)(H(2)O)(2). The observed significant decrease in the rate of NO binding to (P)Fe(OH) as compared to that of (P)Fe(H(2)O)(2) does not conform with expectations based on previous mechanistic work on NO-heme interactions, which would point to a diffusion-limited reaction for the five-coordinate Fe(III) center in (P)Fe(OH). The decrease in rate and an associatively activated mode of NO binding observed at high pH is ascribed to an increase in the activation barrier related to spin state and structural changes accompanying NO coordination to the high-spin (P)Fe(III)(OH) complex. The existence of such a barrier has previously been observed in the reactions of five-coordinate iron(II) hemes with CO and is evidenced for the first time for the process involving coordination of NO to the iron heme complex. The observed reactivity pattern, relevant in the context of studies on NO interactions with synthetic and biologically important hemes (in particular, hemoproteins), is reported here for an example of a simple water-soluble iron(III) porphyrin [meso-tetrakis(sulfonatomesityl)porphinato]-iron(III), (TMPS)Fe(III).     

Comparison of thioethers and sulfoxides as axial ligands for N-acetylmicroperoxidase-8: implications for oxidation of methionine-80 in cytochrome c

Methionine-80 (Met-80) in mitochondrial cytochrome c (cyt c) can be oxidized to the corresponding sulfoxide by reactive oxygen species, a reaction of potential biological significance. As an approach to investigating how oxidation of Met-80 would influence its interactions with heme iron, we have examined binding of 2-(methylthio)ethanol (MTE) and dimethyl sulfoxide (DMSO), models for the side chains of Met and Met(SO), respectively, to ferrous and ferric N-acetylmicroperoxidase-8 (AcMP8). We find that DMSO coordinates 1.2 kcal/mol less strongly to Fe(III)-AcMP8 than does MTE, although both ligands form low-spin complexes. Comparison of spectroscopic data for the DMSO complex of Fe(III)-AcMP8 with published data for the Met(SO)-80 form of ferric cyt c allows us to conclude that Met(SO)-80 does not coordinate to iron in the latter. DMSO coordinates to Fe(II)-AcMP8 1.3 kcal/mol more strongly than does MTE, whereas Met-80 and Met(SO)-80 are reported to have approximately equal affinity for Fe(II) in cyt c. This result suggests that the steric environment near the heme iron in cyt c discriminates against coordination of Met(SO)-80. Vacuum quantum chemical density functional theory calculations confirm the greater affinity of the sulfoxide and show that coordination via oxygen is strongly favored. Resonance Raman spectroscopic data indicate that the preference for coordination via oxygen is maintained in solution. The computational data further indicate that the DMSO complex derives significant enthalpic stabilization from pi back-bonding but that iron to sulfur pi back-bonding does not make a significant contribution to bonding in the thioether complex.     

Amorphous iron-(hydr) oxide networks at liquid/vapor interfaces: In situ X-ray scattering and spectroscopy studies

Surface sensitive X-ray reflectivity (XR), fluorescence (XF), and grazing incidence X-ray diffraction (GIXD) experiments were conducted to determine the accumulation of ferric iron Fe (III) or ferrous iron Fe (II) under dihexadecyl phosphate (DHDP) or arachidic acid (AA) Langmuir monolayers at liquid/vapor interfaces. Analysis of the X-ray reflectivity and fluorescence data of monolayers on the aqueous subphases containing FeCl(3) indicates remarkably high levels of surface-bound Fe (III) in number of Fe(3+) ions per molecule (DHDP or AA) that exceed the amount necessary to neutralize a hypothetically completely deprotonated monolayer (DHDP or AA). These results suggest that nano-scale iron (hydr) oxide complexes (oxides, hydroxides or oxyhydroxides) bind to the headgroups and effectively overcompensate the maximum possible charges at the interface. The lack of evidence of in-plane ordering in GIXD measurements and strong effects on the surface-pressure versus molecular area isotherms indicate that an amorphous network of iron (hydr) oxide complexes contiguous to the headgroups is formed. Similar experiments with FeCl(2) generally resulted with the oxidation of Fe (II)-Fe (III) which consequently leads to ferric Fe (III) complexes binding albeit with less iron at the interface. Controlling the oxidation of Fe (II) changes the nature and amount of binding significantly. The implications to biomineralization of iron (hydr) oxides are briefly discussed.