Lauren E. Goodrich

Electronic structure of heme-nitrosyls and its significance for nitric oxide reactivity, sensing, transport, and toxicity in biological systems.

This review summarizes recent developments in the investigation of the electronic structures, spectroscopic properties, and reactivities of ferrous and ferric heme-nitrosyls and how this relates to important biological processes. Ferrous heme-nitrosyls show interesting variations in electronic structure as a function of the different types of proximal ligands, as is evident from electron paramagnetic resonance, magnetic circular dichroism, and vibrational spectroscopy. In particular, coordination of imidazoles like histidine (His) increases the radical character on NO and, in this way, could help activate the bound NO for catalysis. Vice versa, the bound NO ligand imposes a strong sigma trans effect on the proximal His, which, in the case of soluble guanylate cyclase (sGC), the biological NO sensor protein, induces breaking of the Fe(II)-His bond and activates the protein. The possibility of sGC activation by HNO is also discussed. Finally, the properties of ferrous heme-nitrosyls with proximal cysteinate (Cys) coordination are evaluated. It has been known for some time that ferric heme-nitrosyls are intrinsically more labile than their ferrous counterparts, but the underlying reasons for this observation have not been clarified. New results show that this property relates to the presence of a low-lying excited state that is dissociative with respect to the Fe(III)-NO bond. On the other hand, the ground state of these complexes is best described as Fe(II)-NO(+), which shows a very strong Fe-NO bond, as is evident from vibrational spectroscopy. NO, therefore, is a weak ligand to ferric heme, which, at the same time, forms a strong Fe-NO bond. This is possible because the thermodynamic weakness and spectroscopic strength of the Fe-NO bond relate to the properties of different electronic states. Thiolate coordination to ferric hemes leads to a weakening of both the Fe-NO and N-O bonds as a function of the thiolate donor strength. This observation can be explained by a sigma backbond into the sigma* orbital of the Fe-N-O unit that is mediated by the thiolate sigma-donor orbital via orbital mixing. This is a new interaction in heme-nitrosyl that has not been observed before. This also induces a bending of the Fe-N-O subunit in these cases. New spectroscopic data on a corresponding model complex are included in this paper. Finally, the mechanism of NO reduction by cytochrome P450nor is elucidated based on recent density functional theory results.

Electronic Structure and Biologically Relevant Reactivity of Low-Spin {FeNO}8 Porphyrin Model Complexes: New Insight from a Bis-Picket Fence Porphyrin

Because of HNOs emerging role as an important effector molecule in biology, there is great current interest in the coordination chemistry of HNO and its deprotonated form, the nitroxyl anion (NO(-)), with hemes. Here we report the preparation of four new ferrous heme-nitroxyl model complexes, {FeNO}(8) in the Enemark-Feltham notation, using three electron-poor porphyrin ligands and the bis-picket fence porphyrin H2[3,5-Me-BAFP] (3,5-Me-BAFP(2-) = 3,5-methyl-bis(aryloxy)-fence porphyrin dianion). Electrochemical reduction of [Fe(3,5-Me-BAFP)(NO)] (1-NO) induces a shift of ν(N-O) from 1684 to 1466 cm(-1), indicative of formation of [Fe(3,5-Me-BAFP)(NO)](-) (1-NO(-)), and similar results are obtained with the electron-poor hemes. These results provide the basis to analyze general trends in the properties of ferrous heme-nitroxyl complexes for the first time. In particular, we found a strong correlation between the electronic structures of analogous {FeNO}(7) and {FeNO}(8) complexes, which we analyzed using density functional theory (DFT) calculations. To further study their reactivity, we have developed a new method for the preparation of bulk material of pure heme {FeNO}(8) complexes via corresponding [Fe(porphyrin)](-) species. Reaction of [Fe(To-F2PP)(NO)](-) (To-F2PP(2-) = tetra(ortho-difluorophenyl)porphyrin dianion) prepared this way with acetic acid generates the corresponding {FeNO}(7) complex along with the release of H2. Importantly, this disproportionation can be suppressed when the bis-picket fence porphyrin complex [Fe(3,5-Me-BAFP)(NO)](-) is used, and excitingly, with this system we were able to generate the first ferrous heme-NHO model complex reported to date. The picket fence of the porphyrin renders this HNO complex very stable, with a half-life of ~5 h at room temperature in solution. Finally, with analogous {FeNO}(8) and {FeNHO}(8) complexes in hand, their biologically relevant reactivity toward NO was then explored.

Iron-Porphyrin NO Complexes with Covalently Attached N-Donor Ligands: Formation of a Stable Six-Coordinate Species in Solution

A series of substituted tetraphenylporphyrin type macrocycles (TMP or To-F(2)PP) with covalently attached N-donor ligands (pyridine or imidazole linker) have been synthesized. Linkers with varying chain lengths and designs have been applied to systematically investigate the effect of chain length and rigidity on the binding affinity of the linker to the corresponding Fe(II)-NO heme complexes. The binding of the linker is monitored in solution using a variety of spectroscopic methods including UV-vis absorption, EPR, and IR spectroscopy. Both the N-O stretching frequency and the imidazole (14)N hyperfine coupling constants show a good correlation with the Fe-(N-donor) bond strength in these systems. The complexes with covalently attached pyridyl and alkyl imidazole ligands only exhibit weak interactions of the linker with iron(II). However, the stable six-coordinate complex [Fe(To-F(2)PP-BzIM)(NO)] (4) is obtained when a rigid benzyl linker is applied. This complex exhibits typical properties of six-coordinate ferrous heme-nitrosyls in which an N-donor ligand is bound trans to NO, including the Soret band at 427 nm and the typical nine line (14)N hyperfine splitting in the EPR spectrum. A crystal structure has been obtained for the corresponding zinc complex. Here, we report the first systematic study on the requirements for the formation of stable six-coordinate ferrous heme nitrosyl complexes in solution at room temperature in the absence of excess axial N-donor ligand.

Vibrational Assignments of Six-Coordinate Ferrous Heme Nitrosyls: New Insight From Nuclear Resonance Vibrational Spectroscopy

This Communication addresses a long-standing problem: the exact vibrational assignments of the low-energy modes of the Fe-N-O subunit in six-coordinate ferrous heme nitrosyl model complexes. This problem is addressed using nuclear resonance vibrational spectroscopy (NRVS) coupled to (15)N(18)O isotope labeling and detailed simulations of the obtained data. Two isotope-sensitive features are identified at 437 and 563 cm(-1). Normal coordinate analysis shows that the 437 cm(-1) mode corresponds to the Fe-NO stretch, whereas the 563 cm(-1) band is identified with the Fe-N-O bend. The relative NRVS intensities of these features determine the degree of vibrational mixing between the stretch and the bend. The implications of these results are discussed with respect to the trans effect of imidazole on the bound NO. In addition, a comparison to myoglobin-NO (Mb-NO) is made to determine the effect of the Mb active site pocket on the bound NO.

The trans effect of nitroxyl (HNO) in ferrous heme systems: Implications for soluble guanylate cyclase activation by HNO

Soluble guanylate cyclase (sGC) is the primary mammalian nitric oxide (NO) sensor. Through the strong thermodynamic σ-trans effect of NO, binding of NO at the distal side of the ferrous heme induces cleavage of the proximal FeN(His) bond, activating the catalytic domain of the enzyme. It has been proposed that nitroxyl (HNO) is also capable of activating sGC, but the key question remains as to whether HNO can induce cleavage of the FeN(His) bond. Here we report calculated binding constants for 1-methylimidazole (MI) to [Fe(P)(X)] (P=porphine(2-)) where X=NO, HNO, CO, and MI to evaluate the trans interaction of these groups, X, with the proximal imidazole (histidine) in sGC. Systematic assessment of DFT methods suggests that the prediction of accurate MI binding constants is critically dependent on the inclusion of van der Waals interactions (-D functionals). Calculated (B3LYP-D/TZVP) MI binding constants for X=NO and MI are 110 and 5.6 × 10(5)M(-1), respectively, predicted only one order of magnitude higher than the corresponding experimentally determined values. MI binding constants where X=HNO and CO are consistently predicted to be essentially equal and ~six orders of magnitude larger than those of NO, indicating that CO and HNO mediate a weak thermodynamic trans effect in this system. Orbital analysis of the key σ-bonding orbital, π*(h)_d(z2), and comparison of FeN(MI) bond lengths support this prediction. This suggests that HNO does not induce a σ-trans effect strong enough to promote cleavage of the FeN(His) bond-a key step in the activation of sGC.

Preparation of the Elusive [(por)Fe(NO)(O-ligand)] Complex by Diffusion of Nitric Oxide into a Crystal of the Precursor†

Ferric heme proteins with nitric oxide (NO) as an axial ligand are critical components for the regulation of NO biosynthesis by the enzyme NO synthase, for NO transport (as vasodilator) in nitrophorins, for NO inhibition of cytochrome P450 and related enzymes, and these species have been identified as intermediates in cyt cd1 dissimilatory nitrite reductases and in NO reduction by a fungal cyt P450 NO reductase. Ferric heme nitrosyls are generally described by the Fe NO electronic ground state that is dominated by p backbonding, although the alternative low-spin Fe NO electronic state is close in energy. The physiological functions of ferric nitrosyl heme proteins are clearly dependent on the character of their Fe NO moities. Thus, it is of great interest to investigate a range of ferric nitrosyl hemes that vary in trans axial ligand donor character (for example, S-, N-, and O-donors), as these trans ligands are expected to influence the electronics and hence functions of the Fe NO groups. Previous investigations of the effect of axial trans ligands on ferric NO groups have focused on thiolate systems, and a surprising trans effect of axial thiolate coordination was reported to influence both the geometry and electronics of the Fe NO unit. Unfortunately, ferric nitrosyl hemes are much more difficult to isolate in pure form than their ferrous counterparts owing to the relatively weak binding of NO to ferric heme. This difficulty in the isolation and structural characterization of ferric heme nitrosyls has greatly hindered a complete analysis of the effects of NO binding to these ferric hemes. Heme catalases 10] and heme-binding HasAp proteins contain tyrosine as an axial ligand. The coordination of tyrosine to the heme plays a critical role for the functions that these proteins carry out. It is known that the direct binding of NO to the iron center in heme catalases can result in reversible inhibition of enzyme activity, and the Cat(NO) and Cat(NO) compounds have been reported previously. However, only one protein crystal structure of a catalase–NO derivative has been reported; unfortunately, the low occupancy of NO (ca. 55%) and resolution of the structure prevented an accurate determination of the Fe NO geometry (which could be modeled in the 175–1608 range with weak restraints, and up to ca. 1108 if no restraints were applied). Surprisingly, no neutral [(por)Fe(NO)(O-ligand)] model species has been characterized by spectroscopy or crystallography to model the active site of NO-inhibited heme catalase and related proteins. The closest examples are the structurally characterized [(TPP)Fe(NO)(H2O)]ClO4, [16] [(TPP)Fe(NO)(H2O)]SO3CF3, [17] and [(TPP)Fe(NO)(HO-i-C5H11)]ClO4 species [18]

Five- and Six-Coordinate Adducts of Nitrosamines with Ferric Porphyrins: Structural Models for the Type II Interactions of Nitrosamines with Ferric Cytochrome P450

Nitrosamines are well-known for their toxic and carcinogenic properties. The metabolic activation of nitrosamines occurs via interaction with the heme-containing cytochrome P450 enzymes. We report the preparation and structural characterization of a number of nitrosamine adducts of synthetic iron porphyrins. The reactions of the cations [(por)Fe(THF)(2)]ClO(4) (por = TPP, TTP, OEP) with dialkylnitrosamines (R(2)NNO; R(2) = Me(2), Et(2), (cyclo-CH(2))(4), (cyclo-CH(2))(5), (PhCH(2))(2)) in toluene generate the six-coordinate high-spin (S = 5/2) [(por)Fe(ONNR(2))(2)]ClO(4) compounds and a five-coordinate intermediate-spin (S = 3/2) [(OEP)Fe(ONNMe(2))]ClO(4) derivative in 57-72% yields (TPP = 5,10,15,20-tetraphenylporphyrinato dianion, TTP = 5,10,15,20-tetra-p-tolylporphyrinato dianion, OEP = 2,3,7,8,12,13,17,18-octaethylporphyrinato dianion). The N-O and N-N vibrations of the coordinated nitrosamine groups in [(por)Fe(ONNR(2))(2)]ClO(4) occur in the 1239-1271 cm(-1) range. Three of the six-coordinate [(por)Fe(ONNR(2))(2)]ClO(4) compounds and one five-coordinate [(OEP)Fe(ONNMe(2))]ClO(4) compound have been characterized by single crystal X-ray crystallography. All the nitrosamine ligands in these complexes bind to the ferric centers via a sole eta(1)-O binding mode. No arylnitrosamine adducts were obtained from the reactions of the precursor compounds [(por)Fe(THF)(2)]ClO(4) with three arylnitrosamines (Ph(2)NNO, Ph(Me)NNO, Ph(Et)NNO). However, prolonged exposure of [(por)Fe(THF)(2)]ClO(4) to these arylnitrosamines resulted in the formation of the known five-coordinate (por)Fe(NO) derivatives. The latter (por)Fe(NO) compounds were obtained more readily by the reactions of the three arylnitrosamines with the four-coordinate (por)Fe(II) precursors.

Ligand recruitment and spin transitions in the solid-state photochemistry of Fe(III)TPPCl.

We report evidence for the formation of long-lived photoproducts following excitation of iron(III) tetraphenylporphyrin chloride (Fe(III)TPPCl) in a 1:1 glass of toluene and CH(2)Cl(2) at 77 K. The formation of these photoproducts is dependent on solvent environment and temperature, appearing only in the presence of toluene. No long-lived product is observed in neat CH(2)Cl(2) solvent. A 2-photon absorption model is proposed to account for the power-dependent photoproduct populations. The products are formed in a mixture of spin states of the central iron(III) metal atom. Metastable six-coordinate high-spin and low-spin complexes and a five-coordinate high-spin complex of iron(III) tetraphenylporphyrin are assigned using structure-sensitive vibrations in the resonance Raman spectrum. These species appear in conjunction with resonantly enhanced toluene solvent vibrations, indicating that the Fe(III) compound formed following photoexcitation recruits a toluene ligand from the surrounding environment. Low-temperature transient absorption (TA) measurements are used to explain the dependence of product formation on excitation frequency in this photochemical model. The six-coordinate photoproduct is initially formed in the high-spin Fe(III) state, but population relaxes into both high-spin and low-spin state at 77 K. This is the first demonstration of coupling between the optical and magnetic properties of an iron-centered porphyrin molecule.

The Interaction of HNO With Transition Metal Centers and Its Biological Significance. Insight Into Electronic Structure From Theoretical Calculations

HNO is a biological effector molecule of growing significance that is proposed to act both as a signaling molecule in mammals, but also as a drug that elevates the detrimental effects of heart attack and stroke by preventing reperfusion injuries. Although the endogenous production of HNO has never been shown rigorously, chemists have discovered a number of pathways that nature could use to produce this molecule from NO, many of them involving transition metal centers. In addition, in its potential role as a signaling molecule, transition metal centers, especially heme and nonheme iron sites, are prime candidates as HNO receptors. In this regard, it is also noteworthy that soluble guanylate cyclase has been proposed to be activated by HNO. In this chapter, we elucidate the electronic structure of heme–HNO complexes with a special focus on their potential role in biology. Other recently discovered HNO complexes based on pentacyanoferrate and [Ru II (Me 3 [9]aneN 3 )(bpy)] 2+ are also included. Insight into the electronic structure of all of these HNO complexes is derived from carefully performed quantum-chemical calculations, usually DFT methods, in close correlation to the available experimental (spectroscopic) data. A critical perspective on the successes and failures of DFT in accomplishing this goal is also provided. For this purpose, the ability of different DFT methods to predict (a) accurate binding constants of biologically relevant molecules to ferrous hemes, and (b) the p K a ’s of HNO complexes is evaluated.