Spectroscopic properties and electronic structure of five- and six-coordinate iron(II) porphyrin NO complexes: Effect of the axial N-donor ligand

In this paper, the differences in the spectroscopic properties and electronic structures of five- and six-coordinate iron(II) porphyrin NO complexes are explored using [Fe(TPP)(NO)] (1; TPP = tetraphenylporphyrin) and [Fe(TPP)(MI)(NO)] (2; MI = 1-methylimidazole) type systems. Binding of N-donor ligands in axial position trans to NO to five-coordinate complexes of type 1 is investigated using UV-vis absorption and 1H NMR spectroscopies. This way, the corresponding binding constants Keq are determined and the 1H NMR spectra of 1 and 2 are assigned for the first time. In addition, 1H NMR allows for the determination of the degree of denitrosylation in solutions of 1 with excess base. The influence of the axial ligand on the properties of the coordinated NO is then investigated. Vibrational spectra (IR and Raman) of 1 and 2 are presented and assigned using isotope substitution and normal-coordinate analysis. Obtained force constants are 12.53 (N-O) and 2.98 mdyn/A (Fe-NO) for 1 compared to 11.55 (N-O) and 2.55 mdyn/A (Fe-NO) for 2. Together with the NMR results, this provides experimental evidence that binding of the trans ligand weakens the Fe-NO bond. The principal bonding schemes of 1 and 2 are very similar. In both cases, the Fe-N-O subunit is strongly bent. Donation from the singly occupied pi* orbital of NO into d(z2) of iron(II) leads to the formation of an Fe-NO sigma bond. In addition, a medium-strong pi back-bond is present in these complexes. The most important difference in the electronic structures of 1 and 2 occurs for the Fe-NO sigma bond, which is distinctively stronger for 1 in agreement with the experimental force constants. The increased sigma donation from NO in 1 also leads to a significant transfer of spin density from NO to iron, as has been shown by magnetic circular dichroism (MCD) spectroscopy in a preceding Communication (Praneeth, V. K. K.; Neese, F.; Lehnert, N. Inorg. Chem. 2005, 44, 2570-2572). This is confirmed by the 1H NMR results presented here. Hence, further experimental and computational evidence is provided that complex 1 has noticeable Fe(I)NO+ character relative to 2, which is an Fe(II)NO(radical) complex. Finally, using MCD theory and quantum chemical calculations, the absorption and MCD C-term spectra of 1 and 2 are assigned for the first time.     

Spin density distribution in five- and six-coordinate iron(II)-porphyrin NO complexes evidenced by magnetic circular dichroism spectroscopy

Using magnetic circular dichroism (MCD) spectroscopy together with DFT calculations, the spin density distributions in five-coordinate [Fe(TPP)(NO)] (I) and six-coordinate [Fe(TPP)(MI)(NO)] (II, MI = 1-methylimidazole) are defined. In the five-coordinate complex, a strong Fe-NO sigma bond between pi(*)(h) and d(z)(2) is present that leads to a large transfer of spin density from the NO ligand to Fe(II) corresponding to an electronic structure with noticeable Fe(I)-NO(+) character. Consequently, the MCD spectrum is dominated by paramagnetic C-term contributions. On coordination of the sixth ligand, the spin density is pushed back from the iron toward the NO ligand, resulting in an Fe(II)-NO(radical) type of electronic structure. This is reflected by the fact that the MCD spectrum is dominated by diamagnetic contributions.     

Detection and determination of the {Fe(NO)(2)} core vibrational features in dinitrosyl-iron complexes from experiment, normal coordinate analysis, and density functional theory: an avenue for probing the nitric oxide oxidation state

As it is now well-established that nitric oxide plays an important role in many physiological processes, there is a renewed interest in dinitrosyl-iron complexes (DNICs). The question concerning the electronic structure of DNICs circles around the formal oxidation states of the iron and nitric oxide of the Fe(NO)2 core. Previous infrared measurements of nu(NO) alone point out inconsistencies in assigning electron configurations and charges on metals, inherent from the measurement of one parameter external to the metal. This work represents the first experimental and theoretical attempt to assign vibrational modes for the {Fe(NO)2}9 core of DNICs. The following complexes are investigated, [PPN][S5Fe(NO)2] (1), [PPN][Se5Fe(NO)2] (2), [PPN][(SPh)2Fe(NO)2] (3), and [PPN][(SePh)2Fe(NO)2] (4). The analysis of isotopically edited Raman data together with normal coordinate calculation permitted assignment of nu(NO) and nu(Fe-NO) stretching and delta(Fe-N-O) bending modes in these complexes. The assignments proposed are the first ever reported for the DNICs; a comparison of nu(NO) and nu(Fe-NO) stretching frequencies in DNICs is now feasible. The Fe(NO)2 core electronic configuration in these complexes is described as {Fe1+(*NO)2}. Results from 1 and 3 have been complemented by density functional theory (DFT) frequency calculations. In addition to providing a reasonably correct account of the observed frequencies, DFT calculations also give a good account of the frequency shifts upon 15NO substitution providing the first link between DFT and Raman spectroscopies for DNICs. Through the use of a combination of NO intraligand and metal-ligand vibrational data for the Fe(NO)2 core, normal coordinate analysis gives a NO stretching force constant, which compared to molecular NO gas, is significantly reduced for all four complexes. The hybrid U-B3LYP/6-311++G(3d,2p) density functional method has been employed to analyze the molecular orbital compositions of predominantly NO orbitals based on the crystal structure of complex 1. The molecular orbital not only revealed the bonding nature of the {Fe(NO)2}9 core but also provided a qualitative correct account of the observed low NO vibrational frequencies. The calculation shows that the NO is involved in a strong donor bonding interaction with the Fe1+. This donor bonding interaction involves the 5sigma molecular orbital of the NO, which is sigma-bonding with respect to the intramolecular NO bond, and removal of electron density from this orbital destabilizes the NO bond. Though it is too ambiguous to extrapolate a nu(Fe-NO)/nu(NO) correlation line for {Fe(NO)2}9 DNICs based only on the data reported here, the feasibility of using a vibrational systematics diagram to extract the electron configurations and charges on metals is demonstrated based on the vibrational data available in the literature for iron-nitrosyl complexes. The data provided here can be used as a model for the determination of effective charges on iron and the bonding of nitric oxides to metals in DNICs.     

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.     

Theoretical investigations of the electronic structure and spectroscopy of mononuclear, non-heme [Fe-NO](6) complexes

The unusual metal coordination and spin-state of the Fe(III) center in nitrile hydratase (NHase) has stimulated the synthesis of numerous model complexes in efforts to understand the reactivity and spectroscopic properties of the enzyme. A particular problem has been the development of model Fe(III) complexes that exhibit reversible, photolabile binding to nitric oxide (NO) in a manner similar to that observed for the NHase metal center. We now report a detailed NBO analysis of the ground-state chemical bonding in three [Fe-NO](6) complexes that exhibit different responses to irradiation, together with investigations of their spectroscopic properties using semiempirical INDO/S CI singles calculations. Our computational studies reveal a correlation between the photolability of these complexes and the existence of low-energy transitions that promote an electron into the Fe-NO pi(*) antibonding molecular orbital. In addition to providing detailed insights into how the ligand field influences the spectroscopy of these mononuclear complexes, these studies strengthen our previous conclusions regarding the role of post-translational cysteine modification in modulating the photoreactivity of the inactive, NO complex of NHase.     

Spectroscopic studies of the effect of ligand donor strength on the Fe-NO bond intradiol dioxygenases

The geometric and electronic structure of NO bound to reduced protocatechuate 3,4-dioxygenase and its substrate (3,4-dihydroxybenzoate, PCA) complex have been examined by X-ray absorption (XAS), UV-vis absorption (Abs), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The results are compared to those previously published on model complexes described as [FeNO]7 systems in which an S = 5/2 ferric center is antiferromagnetically coupled to an S = 1 NO-. XAS pre-edge analysis indicates that the Fe-NO units in FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] lack the greatly increased pre-edge intensity representative of most [FeNO]7 model sites. Furthermore, from extended X-ray absorption fine structure (EXAFS) analysis, the FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] active sites are shown to have an Fe-NO distance of at least 1.91 A, approximately 0.2 A greater than those found in the model complexes. The weakened Fe-NO bond is consistent with the overall lengthening of the bond lengths and the fact that VTVH MCD data show that NO(-)-->FeIII CT transitions are no longer polarized along the z-axis of the zero-field splitting tensor. The weaker Fe-NO bond derives from the strong donor interaction of the endogenous phenolate and substrate catecholate ligands, which is observed from the increased intensity in the CT region relative to that of [FeNO]7 model complexes, and from the shift in XAS edge position to lower energy. As NO is an analogue of O2, the effect of endogenous ligand donor strength on the Fe-NO bond has important implications with respect to O2 activation by non-heme iron enzymes.     

Spectroscopic and computational investigation of second-sphere contributions to redox tuning in Escherichia coli iron superoxide dismutase

In Fe- and Mn-dependent superoxide dismutases (SODs), second-sphere residues have been implicated in precisely tuning the metal ion reduction potential to maximize catalytic activity (Vance, C. K.; Miller, A.-F. J. Am. Chem. Soc. 1998, 120, 461-467). In the present study, spectroscopic and computational methods were used to characterize three distinct Fe-bound SOD species that possess different second-coordination spheres and, consequently, Fe(3+/2+)reduction potentials that vary by approximately 1 V, namely, FeSOD, Fe-substituted MnSOD (Fe(Mn)SOD), and the Q69E FeSOD mutant. Despite having markedly different metal ion reduction potentials, FeSOD, Fe(Mn)SOD, and Q69E FeSOD exhibit virtually identical electronic absorption, circular dichroism, and magnetic circular dichroism (MCD) spectra in both their oxidized and reduced states. Likewise, variable-temperature, variable-field MCD data obtained for the oxidized and reduced species do not reveal any significant electronic, and thus geometric, variations within the Fe ligand environment. To gain insight into the mechanism of metal ion redox tuning, complete enzyme models for the oxidized and reduced states of all three Fe-bound SOD species were generated using combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations. Consistent with our spectroscopic data, density functional theory computations performed on the corresponding active-site models predict that the three SOD species share similar active-site electronic structures in both their oxidized and reduced states. By using the QM/MM-optimized active-site models in conjunction with the conductor-like screening model to calculate the proton-coupled Fe(3+/2+) reduction potentials, we found that different hydrogen-bonding interactions with the conserved second-sphere Gln (changed to Glu in Q69E FeSOD) greatly perturb the p K of the Fe-bound solvent ligand and, thus, drastically affect the proton-coupled metal ion reduction potential.     

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.     

二甲胺基尾式卟啉铁与双原子小分子CO,NO配位性质的研究

本文报道了二甲胺基尾式卟啉铁{中位-[邻(4-二甲胺基丁酰胺基)苯基]三苯基卟啉合铁(II)}同双原子小分子CO, NO的配合物的UV, MCD, ESR光谱和电化学性质, 测定了它与CO轴向加合平衡常数为1.38x10^7M^-^1, 讨论了尾端配体对CO与中心离子铁的成键影响。NO配合物的ESR和循环伏安结果表明, NO的配位削弱了尾端N与中心离子铁的键强, 并用分子轨道理论解释了这一结果。     

A density functional theory investigation of Fe-N-O bonding in heme proteins and model systems

We report the results of a series of density functional theory (DFT) calculations of the M?ssbauer quadrupole splittings and isomer shifts in NO heme model compounds, together with the results of calculations of the M?ssbauer quadrupole splittings, isomer shifts, and electron paramagnetic resonance hyperfine coupling constants in a model Fe(II)(NO)(imidazole) complex as a function of Fe-NO bond length and Fe-N-O bond angle. The results of the M?ssbauer quadrupole splitting and isomer shift calculations on the NO heme model compounds show good accord between theory and experiment, with the largest errors being observed for structures having the largest crystallographic R(1) values. The results of the property surface calculations were then used to calculate Fe-NO bond length and Fe-N-O bond angle probability surfaces (Z-surfaces) for a nitrosyl hemoglobin, using, in addition, an energy filter. The results obtained yielded a most probable Fe-NO bond length (r) of 1.79 A and an Fe-N-O bond angle (beta) of 136 degrees -137 degrees. This bond length is somewhat longer than those observed in most model compounds but may be due, at least in part, to hydrogen bond formation with the distal His residue. Bond elongation was also observed in a geometry optimized Fe(II)(NO)(imidazole) complex hydrogen bonded to an imidazole residue, in which we find r = 1.76-1.78 A and beta = 137 degrees -138 degrees. The computed bond angles are close to the canonical approximately 140 degrees value found in most model systems. Highly bent Fe-N-O bond angles or very long Fe-NO bond lengths seem unlikely to occur in proteins, due to their high energies. We also investigated the molecular orbitals and spin densities in each of the six coordinate systems investigated and found the orbitals and spin densities to be generally similar those described previously for five coordinate systems. Taken together, these results show that M?ssbauer quadrupole splittings and isomer shifts, in addition to electron paramagnetic resonance hyperfine coupling constants, can now be calculated for nitrosyl heme systems with relatively good accuracy and that the results so obtained can be used to determine Fe-N-O geometries in metalloproteins. The Z-surface approach is thus applicable to both diamagnetic (CO) and paramagnetic (NO) heme proteins with in both cases the metal-ligand binding geometries found in the proteins being very close to those seen in model systems.     

Influence of thiolate ligands on reductive N-O bond activation. Probing the O2(-) binding site of a biomimetic superoxide reductase analogue and examining the proton-dependent reduction of nitrite

Nitric oxide (NO) is frequently used to probe the substrate-binding site of "spectroscopically silent" non-heme Fe(2+) sites of metalloenzymes, such as superoxide reductase (SOR). Herein we use NO to probe the superoxide binding site of our thiolate-ligated biomimetic SOR model [Fe(II)(S(Me(2))N(4)(tren))](+) (1). Like NO-bound trans-cysteinate-ligated SOR (SOR-NO), the rhombic S = 3/2 EPR signal of NO-bound cis-thiolate-ligated [Fe(S(Me(2))N(4)(tren)(NO)](+) (2; g = 4.44, 3.54, 1.97), the isotopically sensitive ν(NO)(ν((15)NO)) stretching frequency (1685(1640) cm(-1)), and the 0.05 ? decrease in Fe-S bond length are shown to be consistent with the oxidative addition of NO to Fe(II) to afford an Fe(III)-NO(-) {FeNO}(7) species containing high-spin (S = 5/2) Fe(III) antiferromagnetically coupled to NO(-) (S = 1). The cis versus trans positioning of the thiolate does not appear to influence these properties. Although it has yet to be crystallographically characterized, SOR-NO is presumed to possess a bent Fe-NO similar to that of 2 (Fe-N-O = 151.7(4)°). The N-O bond is shown to be more activated in 2 relative to N- and O-ligated {FeNO}(7) complexes, and this is attributed to the electron-donating properties of the thiolate ligand. Hydrogen-bonding to the cysteinate sulfur attenuates N-O bond activation in SOR, as shown by its higher ν(NO) frequency (1721 cm(-1)). In contrast, the ν(O-O) frequency of the SOR peroxo intermediate and its analogues is not affected by H-bonds to the cysteinate sulfur or other factors influencing the Fe-SR bond strength; these only influence the ν(Fe-O) frequency. Reactions between 1 and NO(2)(-) are shown to result in the proton-dependent heterolytic cleavage of an N-O bond. The mechanism of this reaction is proposed to involve both Fe(II)-NO(2)(-) and {FeNO}(6) intermediates similar to those implicated in the mechanism of NiR-promoted NO(2)(-) reduction.     

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.     

Electronic structure and reactivity of low-spin Fe(III)-hydroperoxo complexes: comparison to activated bleomycin

The spectroscopic properties, electronic structure, and reactivity of the low-spin Fe(III)-hydroperoxo complex [Fe(N4Py)(OOH)](2+) (1, N4Py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) are investigated in comparison to those of activated bleomycin (ABLM). Complex 1 is characterized by Raman features at 632 (Fe-O stretch) and 790 cm(-1) (O-O stretch), corresponding to a strong Fe-O bond (force constant 3.62 mdyn/A) and a weak O-O bond (3.05 mdyn/A). The UV-vis spectrum of 1 shows a broad absorption band around 550 nm that is assigned to a charge-transfer transition from the hydroperoxo to a t(2g) d orbital of Fe(III) using resonance Raman and MCD spectroscopies and density functional (DFT) calculations. Compared to low-spin [Fe(TPA)(OH(x))(OO(t)Bu)](x+)(TPA = tris(2-pyridylmethyl)amine, x = 1 or 2), an overall similar Fe-OOR bonding results for low-spin Fe(III)-alkylperoxo and -hydroperoxo species. Correspondingly, both systems show similar reactivities and undergo homolytic cleavage of the O-O bond. From the DFT calculations, this reaction is more endothermic for 1 due to the reduced stabilization of the .OH radical compared to .O(t)Bu and the absence of the hydroxo ligand that helps to stabilize the resulting Fe(IV)=O species. In contrast, ABLM has a somewhat different electronic structure where no pi donor bond between the hydroperoxo ligand and iron(III) is present [Neese, F.; Zaleski, J. M.; Loeb-Zaleski, K.; Solomon, E. I. J. Am. Chem. Soc. 2000, 122, 11703]. Possible reaction pathways for ABLM are discussed in relation to known experimental results.     

Unusual role of solvents in the syntheses of {Fe-NO}6,7 nitrosyls derived from a ligand with carboxamido nitrogen and thiolato sulfur donors

Reaction of excess NO with the S = 3/2 Fe(III) complex (Et4N)2[Fe(PhPepS)(Cl)] (1) in protic solvents such as MeOH affords the {Fe-NO}(7) nitrosyl (Et(4)N)(2)[Fe(PhPepS)(NO)] (2). This distorted square-pyramidal S = 1/2 complex, a product of reductive nitrosylation, is the first example of an {Fe-NO}7 nitrosyl with carboxamido-N and thiolato-S coordination. When the same reaction is performed in aprotic solvents such as MeCN and DMF, the product is a dimeric diamagnetic {Fe-NO}6 complex, (Et4N)2-[{Fe(PhPepS)(NO)}2] (3). Both electrochemical and chemical oxidation of 2 leads to the formation of 3 via a transient five-coordinate {Fe-NO}6 intermediate. The oxidation is NO-centered. The ligand frame is not attacked by excess NO in these reactions.     

Spectroscopic and computational insights into second-sphere amino-acid tuning of substrate analogue/active-site interactions in iron(III) superoxide dismutase

In this study, the mechanism by which second-sphere residues modulate the structural and electronic properties of substrate-analogue complexes of the Fe-dependent superoxide dismutase (FeSOD) has been explored. Both spectroscopic and computational methods were used to investigate the azide (N3(-)) adducts of Fe(3+)SOD (N3-Fe(3+)SOD) and its Q69E mutant, as well as Fe(3+)-substituted MnSOD (N3-Fe(3+)(Mn)SOD) and its Y34F mutant. Electronic absorption, circular dichroism, and magnetic circular dichroism spectroscopic data reveal that the energy of the dominant N3(-)-->Fe(3+) ligand-to-metal charge transfer (LMCT) transition decreases in the order N3-Fe(3+)(Mn)SOD>N3-Fe(3+)SOD>Q69E N3-Fe(3+)SOD. Intriguingly, the LMCT transition energies correlate almost linearly with the Fe(3+/2+) reduction potentials of the corresponding Fe(3+)-bound SOD species in the absence of azide, which span a range of approximately 1 V (see the preceding paper). To explore the origin of this correlation, combined quantum mechanics/molecular mechanics (QM/MM) geometry optimizations were performed on complete enzyme models. The INDO/S-CI computed electronic transition energies satisfactorily reproduce the experimental trend in LMCT transition energies, indicating that the QM/MM optimized active-site models are reasonable. Density functional theory calculations on these experimentally validated active-site models reveal that the differences in spectral and electronic properties among the four N 3(-) adducts arise primarily from differences in the hydrogen-bond network involving the conserved second-sphere Gln (mutated to Glu in Q69E FeSOD) and the solvent ligand. The implications of our findings with respect to the mechanism by which the second-coordination sphere modulates substrate-analogue binding as well as the catalytic properties of FeSOD are discussed.     

EPR and UV-vis studies of the nitric oxide adducts of bacterial phenylalanine hydroxylase: effects of cofactor and substrate on the iron environment

Phenylalanine hydroxylase from Chromobacterium violaceum (cPAH), which catalyzes phenylalanine oxidation to tyrosine, is homologous to the catalytic domain of eukaryotic PAHs. Previous crystallographic and spectroscopic studies on mammalian PAH conflict on whether O2 binds to the open-coordination site or displaces the remaining water ligand to yield either a six- or a five-coordinate iron, respectively. The abilities of nitric oxide to behave as an oxygen mimic and a spectroscopic probe of ferrous iron are used to investigate the geometric and electronic effects of cofactor and substrate binding to cPAH by electron paramagnetic resonance (EPR) and UV-vis spectroscopies. A rhombic distortion observed for the ternary complex is due to two factors: a decrease in the Fe-NO angle and an alteration in the equatorial ligand geometry. Both factors are consistent with NO displacing the sole remaining water ligand to yield a five-coordinate iron center. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched water is observed in the absence of substrates or presence of cofactor only (binary complex), demonstrating that water is bound to the Fe(II). However, in the presence of substrate and cofactor (ternary complex), the EPR resonances of the nitrosyl complex are not broadened by 17O-enriched water, indicating the displacement of water by NO to afford a five-coordinate iron. Furthermore, the increased intensity in the 500-600 nm range of the UV-vis spectrum of the ternary nitrosyl complex indicates an increased overlap between the in-plane NO 2pi and d(x2-y2) and d(xz) orbitals, which corroborates a five-coordinate iron.     

Unexpected nitrosyl-group bending in six-coordinate [M(NO)](6) sigma-bonded aryl(iron) and -(ruthenium) porphyrins.

The six-coordinate nitrosyl sigma-bonded aryl(iron) and -(ruthenium) porphyrin complexes (OEP)Fe(NO)(p-C(6)H(4)F) and (OEP)Ru(NO)(p-C(6)H(4)F) (OEP = octaethylporphyrinato dianion) have been synthesized and characterized. Single-crystal X-ray structure determinations reveal an unprecedented bending and tilting of the MNO group for both [MNO](6) species as well as significant lengthening of trans axial bond distances. In (OEP)Fe(NO)(p-C(6)H(4)F) the Fe-N-O angle is 157.4(2) degrees, the nitrosyl nitrogen atom is tilted off of the normal to the heme plane by 9.2 degrees, Fe-N(NO) = 1.728(2) A, and Fe-C(aryl) = 2.040(3) A. In (OEP)Ru(NO)(p-C(6)H(4)F) the Ru-N-O angle is 154.9(3) degrees, the nitrosyl nitrogen atom is tilted off of the heme normal by 10.8 degrees, Ru-N(NO) = 1.807(3) A, and Ru-C(aryl) = 2.111(3) A. We show that these structural features are intrinsic to the molecules and are imposed by the strongly sigma-donating aryl ligand trans to the nitrosyl. Density functional-based calculations reproduce the structural distortions observed in the parent (OEP)Fe(NO)(p-C(6)H(4)F) and, combined with the results of extended Hückel calculations, show that the observed bending and tilting of the FeNO group indeed represent a low-energy conformation. We have identified specific orbital interactions that favor the unexpected bending and tilting of the FeNO group. The aryl ligand also affects the Fe-NO pi-bonding as measured by infrared and (57)Fe M?ssbauer spectroscopies. The solid-state nitrosyl stretching frequencies for the iron complex (1791 cm(-)(1)) and the ruthenium complex (1773 cm(-)(1)) are significantly reduced compared to their respective [MNO](6) counterparts. The M?ssbauer data for (OEP)Fe(NO)(p-C(6)H(4)F) yield the quadrupole splitting parameter +0.57 mm/s and the isomer shift 0.14 mm/s at 4.2 K. The results of our study show, for the first time, that bent Fe-N-O linkages are possible in formally ferric nitrosyl porphyrins.