Ferrous NO Research Articles

{FeNO}7 -Type Halogenido Nitrosyl Ferrates: Syntheses, Bonding, and Photoinduced Linkage Isomerism.

Mononitrosyl-iron compounds (MNICs) of the Enemark-Feltham {FeNO}7 type can be divided into a doublet (S=1/2) and a quartet (S=3/2) spin variant. The latter relies on weak-field co-ligands such as amine carboxylates. Aqua-only co-ligation appears to exist in the long-known "brown-ring" [Fe(H2 O)5 (NO)]2+ cation, which was prepared originally from ferrous salts and NO in sulfuric acid. A chloride variant of this species, the green [FeCl3 (NO)]- ion, was first prepared analoguosly by using hydrochloric instead of sulfuric acid. As a tetrahedral species, it is the simple prototype of sulfur-bonded {FeNO}7 (S=3/2) MNICs of biological significance. Although it has been investigated for more than a century, neither clean preparative routes nor reliable structural parameters were available for the [FeCl3 (NO)]- ion and related species such as the [FeCl2 (NO)2 ]- ion, a prototypical dinitrosyliron species (a "DNIC"). In this work, both issues have been resolved. In addition, we report on a computational study on the ground- and excited-state properties including an assignment of the chromophoric transitions. Photoinduced metastable isomers were characterised in a combined experimental and computational approach that resulted in the confirmation of a single photoinduced linkage isomer of the paramagnetic nitrosyl-metal coordination entity.

Non-heme High-Spin {FeNO}6-8 Complexes: One Ligand Platform Can Do It All.

Heme and non-heme iron-nitrosyl complexes are important intermediates in biology. While there are numerous examples of low-spin heme iron-nitrosyl complexes in different oxidation states, much less is known about high-spin (hs) non-heme iron-nitrosyls in oxidation states other than the formally ferrous NO adducts ({FeNO}7 in the Enemark-Feltham notation). In this study, we present a complete series of hs-{FeNO}6-8 complexes using the TMG3tren coligand. Redox transformations from the hs-{FeNO}7 complex [Fe(TMG3tren)(NO)]2+ to its {FeNO}6 and {FeNO}8 analogs do not alter the coordination environment of the iron center, allowing for detailed comparisons between these species. Here, we present new MCD, NRVS, XANES/EXAFS, and Mössbauer data, demonstrating that these redox transformations are metal based, which allows us to access hs-Fe(II)-NO-, Fe(III)-NO-, and Fe(IV)-NO- complexes. Vibrational data, analyzed by NCA, directly quantify changes in Fe-NO bonding along this series. Optical data allow for the identification of a "spectator" charge-transfer transition that, together with Mössbauer and XAS data, directly monitors the electronic changes of the Fe center. Using EXAFS, we are also able to provide structural data for all complexes. The magnetic properties of the complexes are further analyzed (from magnetic Mössbauer). The properties of our hs-{FeNO}6-8 complexes are then contrasted to corresponding, low-spin iron-nitrosyl complexes where redox transformations are generally NO centered. The hs-{FeNO}8 complex can further be protonated by weak acids, and the product of this reaction is characterized. Taken together, these results provide unprecedented insight into the properties of biologically relevant non-heme iron-nitrosyl complexes in three relevant oxidation states.

Structural and Spectroscopic Characterization of a High‐Spin {FeNO}6 Complex with an Iron(IV)−NO− Electronic Structure

AbstractAlthough the interaction of low‐spin ferric complexes with nitric oxide has been well studied, examples of stable high‐spin ferric nitrosyls (such as those that could be expected to form at typical non‐heme iron sites in biology) are extremely rare. Using the TMG3tren co‐ligand, we have prepared a high‐spin ferric NO adduct ({FeNO}6 complex) via electrochemical or chemical oxidation of the corresponding high‐spin ferrous NO {FeNO}7 complex. The {FeNO}6 compound is characterized by UV/Visible and IR spectroelectrochemistry, Mössbauer and NMR spectroscopy, X‐ray crystallography, and DFT calculations. The data show that its electronic structure is best described as a high‐spin iron(IV) center bound to a triplet NO− ligand with a very covalent iron−NO bond. This finding demonstrates that this high‐spin iron nitrosyl compound undergoes iron‐centered redox chemistry, leading to fundamentally different properties than corresponding low‐spin compounds, which undergo NO‐centered redox transformations.

Structural and Spectroscopic Characterization of a High-Spin {FeNO}(6) Complex with an Iron(IV)-NO(-) Electronic Structure.

Although the interaction of low-spin ferric complexes with nitric oxide has been well studied, examples of stable high-spin ferric nitrosyls (such as those that could be expected to form at typical non-heme iron sites in biology) are extremely rare. Using the TMG3 tren co-ligand, we have prepared a high-spin ferric NO adduct ({FeNO}(6) complex) via electrochemical or chemical oxidation of the corresponding high-spin ferrous NO {FeNO}(7) complex. The {FeNO}(6) compound is characterized by UV/Visible and IR spectroelectrochemistry, Mössbauer and NMR spectroscopy, X-ray crystallography, and DFT calculations. The data show that its electronic structure is best described as a high-spin iron(IV) center bound to a triplet NO(-) ligand with a very covalent iron-NO bond. This finding demonstrates that this high-spin iron nitrosyl compound undergoes iron-centered redox chemistry, leading to fundamentally different properties than corresponding low-spin compounds, which undergo NO-centered redox transformations.

Vibrational relaxation of NO stretching modes in ferrous NO and ferric NO in model heme

Femtosecond IR-pump–IR-probe spectroscopy was used to measure the vibrational lifetimes (T1) of NO stretching modes of ferrous NO near 1600cm−1 and ferric NO near 1900cm−1 at room temperature. The T1 of NO bound to the heme, ranging from 3.5 to 34ps, is much shorter in ferrous NO. The vibrational relaxation (VR) of NO was independent of solvent used and excess imidazole concentration, suggesting that intramolecular VR into the internal vibrational modes of the probed molecule may be the dominant pathway for VR of the bound NO. With estimated T1 of the bound NO, we simulated transient spectra of NO bound to ferrous hemoglobin (HbII) after photodeligation of HbIINO and discussed the influence of the hot band on the determination of the dynamics of geminate rebinding of NO to HbII using the change in the magnitude of the fundamental band.

Tetrahydrobiopterin and electron transfer in NO synthase

Mammalian NO synthase requires the cofactor tetrahydrobiopterin (H4B) to act as an electron donor during the activation of molecular oxygen at the heme site. After donating an electron, the resultant H4B radical is then required to abstract an electron from the ferrous NO complex, which is generated at the end of the catalytic reaction, in order to facilitate NO release. We have recently explored the structural requirements of NO synthase for the H4B cofactor by studying a range of novel cofactor analogues with highly modified structures. Substituents on the C6 and C7 positions of H4B are tolerated well, with surprisingly bulky pterins being able to bind and drive NO synthesis. The modified pterins have a wide range of activities and binding constants, but the main function of the cofactors in activating molecular oxygen appears to be independent of C6 and C7 modification as shown by rapid reaction studies. We have also assessed the possibility of direct electron transfer across the dimer interface between H4B molecules in the two NO synthase subunits. The H4B cofactors are within the range for facile electron transfer and present a possible mechanism for NO synthase to escape from the unreactive ferrous-NO complex, which is known to originate from product inhibition.

Oriented single-crystal nuclear resonance vibrational spectroscopy of [Fe(TPP)(MI)(NO)]: quantitative assessment of the trans effect of NO.

This paper presents oriented single-crystal Nuclear Resonance Vibrational Spectroscopy (NRVS) data for the six-coordinate (6C) ferrous heme-nitrosyl model complex [(57)Fe(TPP)(MI)(NO)] (1; TPP(2-) = tetraphenylporphyrin dianion; MI = 1-methylimidazole). The availability of these data enables for the first time the detailed simulation of the complete NRVS data, including the porphyrin-based vibrations, of a 6C ferrous heme-nitrosyl, using our quantum chemistry centered normal coordinate analysis (QCC-NCA). Importantly, the Fe-NO stretch is split by interaction with a porphyrin-based vibration into two features, observed at 437 and 472 cm(-1). The 437 cm(-1) feature is strongly out-of-plane (oop) polarized and shows a (15)N(18)O isotope shift of 8 cm(-1) and is therefore assigned to nu(Fe-NO). The admixture of Fe-N-O bending character is small. Main contributions to the Fe-N-O bend are observed in the 520-580 cm(-1) region, distributed over a number of in-plane (ip) polarized porphyrin-based vibrations. The main component, assigned to delta(ip)(Fe-N-O), is identified with the feature at 563 cm(-1). The Fe-N-O bend also shows strong mixing with the Fe-NO stretching internal coordinate, as evidenced by the oop NRVS intensity in the 520-580 cm(-1) region. Very accurate normal mode descriptions of nu(Fe-NO) and delta(ip)(Fe-N-O) have been obtained in this study. These results contradict previous interpretations of the vibrational spectra of 6C ferrous heme-nitrosyls where the higher energy feature at approximately 550 cm(-1) had usually been associated with nu(Fe-NO). Furthermore, these results provide key insight into NO binding to ferrous heme active sites in globins and other heme proteins, in particular with respect to (a) the effect of hydrogen bonding to the coordinated NO and (b) changes in heme dynamics upon NO coordination. [Fe(TPP)(MI)(NO)] constitutes an excellent model system for ferrous NO adducts of myoglobin (Mb) mutants where the distal histidine (His64) has been removed. Comparison to the reported vibrational data for wild-type (wt) Mb-NO then shows that the effect of H bonding to the coordinated NO is weak and mostly leads to a polarization of the pi/pi* orbitals of bound NO. In addition, the observation that delta(ip)(Fe-N-O) does not correlate well with nu(N-O) can be traced back to the very mixed nature of this mode. The Fe-N(imidazole) stretching frequency is observed at 149 cm(-1) in [Fe(TPP)(MI)(NO)], and spectral changes upon NO binding to five-coordinate ferrous heme active sites are discussed. The obtained high-quality force constants for the Fe-NO and N-O bonds of 2.57 and 11.55 mdyn/A can further be compared to those of corresponding 5C species, which allows for a quantitative analysis of the sigma trans interaction between the proximal imidazole (His) ligand and NO. This is key for the activation of the NO sensor soluble guanylate cyclase. Finally, DFT methods are calibrated against the experimentally determined vibrational properties of the Fe-N-O subunit in 1. DFT is in fact incapable of reproducing the vibrational energies and normal mode descriptions of the Fe-N-O unit well, and thus, DFT-based predictions of changes in vibrational properties upon heme modification or other perturbations of these 6C complexes have to be treated with caution.

Production Of Bioactive NO From The Reaction Of Met-nitrite Hemoglobin With NO: Use Of Glassy Matices And Sol-gels

There is growing evidence that nitrite-mediated reactions of Hb can generate bioactive forms of nitric oxide. The nitrite reductase reaction in which deoxy Hb reacts with nitrite has been studied from this perspective; however, this reaction generates NO under conditions where it can be easily scavenged. In contrast the proposed reaction of NO with nitrite bound to ferric heme derivatives to generate N2O3 is a much more promising mechanism. This follows because N2O3 can rapidly react with thiol containing peptides such as glutathione to produce S-NO derivatives. These species are likely to be the relatively long lived bioactive forms of NO that produce positive physiological effects in the vascular endothelium as occurs for blood substitutes derived from PEGylated Hb. We present results obtained using Hb samples encased in either glassy matrices or sol-gels that directly support this model. In particular there is spectroscopic evidence for the formation of a ferrous intermediate resulting from the direct reaction of NO with met-nitrite derivatives that precedes the formation of either ferrous NO or ferrous CO derivatives. This intermediate is attributable to a ferrous N2O3 complex.

Dinitrosyl–iron complexes with thiol-containing ligands: Spatial and electronic structures

Parameters of the EPR signals of monomeric dinitrosyl–iron complexes with 1 H-1,2,4-triazole-3-thiol (DNIC–MT), obtained by treating MT + ferrous iron in DMSO solution with gaseous NO, have been compared with those of the crystalline monomeric DNIC–MT with tetrahedral structure. Dissolved DNIC–MT were characterized by the isotropic EPR signal centered at g = 2.03 with half-width of 0.7 mT and quintet hyperfine structure when recorded at ambient temperature or the anisotropic EPR signal with g ⊥ = 2.045, g ∥ = 2.014 from frozen solution at 77к. DNIC–MT in crystalline state showed the structure-less symmetrical singlet EPR signal centered at g = 2.03 and half-width of 1.7 mT at both room and liquid nitrogen temperature. The Lorentz shape of this signal indicates the strong exchange interaction between these complexes in the DNIC–MT crystal. Being dissolved in DMSO the crystalline sample of DNIC–MT demonstrated the EPR signal typical for DNIC–MT, obtained by treating MT + ferrous iron in DMSO solution with gaseous NO. Low spin ( S = 1/2) d 9 electron configuration of DNIC–MT with tetrahedral structure (formula {(MT-S ·) 2Fe −1(NO +) 2} +) was suggested to be responsible for the signal of DNIC–MT in crystalline state. Dissolving of the crystals of DNIC–MT may result in the change of their spatial and electronic structure, namely, tetrahedral structure of the complexes characterized by low spin d 9 electronic configuration transforms into a plane-square structure with d 7 electronic configuration and low spin S = 1/2 state (formula {(MT- S −) 2Fe +(NO +) 2} +). The latter was suggested to be characteristic of other DNICs with various thiol-containing ligands in the solutions. The proposed mechanism of these DNICs formation from ferrous iron, thiol and NO shows that the process could be accompanied by the ionization of NO molecules to NO + and NO − ions in the complexes. Detailed analysis of the shape of the EPR signals of these complexes provided additional information about the exchange interaction typical for DNIC–MT in crystals.

Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

Amphitrite ornata dehaloperoxidase (DHP) and Notomastus lobatus chloroperoxidase (NCPO) catalyze the peroxide-dependent dehalogenation of halophenols and halogenation of phenols, respectively. Both enzymes have histidine (His) as their proximal heme iron ligand. Crystallographic examination of DHP revealed that it has a globin fold [M.W. LaCount, E. Zhang, Y.-P. Chen, K. Han, M.M. Whitton, D.E. Lincoln, S.A. Woodin, L. Lebioda, J. Biol. Chem. 275 (2000) 18712–18716] and kinetics studies established that ferric DHP is the active state [R.L. Osborne, L.O. Taylor, K. Han, B. Ely, J.H. Dawson, Biochem. Biophys. Res. Commun. 324 (2004) 1194–1198]. NCPO likely has these same properties. Previous work with His-ligated heme proteins has revealed characteristic spectral distinctions between dioxygen binding globins and peroxide-activating peroxidases. Since DHP, and likely NCPO, is a peroxide-activating globin, we have sought to determine in the present investigation whether the ferric resting states of these two novel heme-containing enzymes are myoglobin-like or peroxidase-like. To do so, we have examined their exogenous ligand-free ferric states as well as their azide, imidazole and NO bound ferric adducts (and ferrous–NO complexes) with UV–Visible absorption and magnetic circular dichroism spectroscopy. We have also compared each derivative to the analogous states of horse heart myoglobin (Mb) and horseradish peroxidase (HRP). The spectra observed for parallel forms of DHP and NCPO are virtually identical to each other as well as to the spectra of the same Mb states, while being less similar to the spectra of corresponding HRP derivatives. From these data, we conclude that exogenous ligand-free ferric DHP and NCPO are six-coordinate with water and neutral His as ligands. This coordination structure is distinctly different from the ferric resting state of His-ligated peroxidases and indicates that DHP and NCPO do not activate bound peroxide through a mechanism dependent on a push effect imparted by a partially ionized proximal His as proposed for typical heme peroxidases.

Regulation of the Monomer-Dimer Equilibrium in Inducible Nitric-oxide Synthase by Nitric Oxide

The oxygenase domain of inducible nitric-oxide synthase exists as a functional tight homodimer in the presence of the substrate L-arginine and the cofactor tetrahydrobiopterin (H4B). In the absence of H4B, the enzyme is a mixture of monomer and loose dimer. We show that exposure of H4B-free enzyme to NO induces dissociation of the loose dimer into monomers in a reaction that follows single exponential decay kinetics with a lifetime of approximately 300 min. It is followed by a faster autoreduction reaction of the heme iron with a lifetime of approximately 30 min and the concurrent breakage of the proximal iron-thiolate bond, forming a five-coordinate NO-bound ferrous species. Mass spectrometry revealed that the NO-induced monomerization is associated with intramolecular disulfide bond formation between Cys104 and Cys109, located in the zinc-binding motif. The regulatory effect of NO as a dimer inhibitor is discussed in the context of the structure/function relationships of this enzyme.

Thiolate coordination to Fe(II)–porphyrin NO centers

The interaction of the Fe(II)–porphyrin NO model complex [Fe(TPP)(NO)] ( 1, TPP = tetraphenylporphyrin) with thiophenolate ligands and tetrahydrothiophene is explored both computationally and experimentally. Complex 1 is reacted with substituted thiophenolates and the obtained six-coordinate adducts of type [Fe(TPP)(SR)(NO)] − are investigated in solution using electron paramagnetic resonance (EPR) spectroscopy. From the obtained g values and 14N hyperfine pattern of the NO ligand it is concluded that the interaction of the thiophenolates with the Fe(II) center is weak in comparison to the corresponding 1-methylimidazole adduct. The strength of the Fe–S bond is increased when alkylthiolates are used as evidenced by comparison with the published EPR spectra of ferrous NO adducts in cytochromes P450 and P450nor, which have an axial cysteinate ligand. These results are further evaluated by density functional (DFT) calculations. The six-coordinate model complex [Fe(P)(SMe)(NO)] − ( 1-SMe; P = porphine ligand used for the calculations) has an interesting electronic structure where NO acts as a medium strong σ donor and π acceptor ligand. Compared to the N-donor adducts with 1-methylimidazole (1-MeIm), etc., donation from the π h ∗ orbital of NO to Fe(II) is reduced due to the stronger trans effect of the alkylthiolate ligand. This is reflected by the predicted longer Fe–NO bond length and smaller Fe–NO force constant for 1-SMe compared to the 1-MeIm adduct. Therefore, the Fe(II)–porphyrin NO adducts with trans alkylthiolate coordination have to be described as Fe(II)–NO(radical) systems. The N–O stretching frequency of these complexes is predicted below 1600 cm −1 in agreement with the available experimental data. In addition, 1-SMe has a unique spin density distribution where Fe has a negative spin density of −0.26 from the calculations. The implications of this unusual electronic structure for the reactivity of the Fe(II)–NO alkylthiolate adducts as they occur in cytochrome P450nor are discussed.

Thermodynamic and Kinetic Analysis of the Nitrosyl, Carbonyl, and Dioxy Heme Complexes of Neuronal Nitric-oxide Synthase: THE ROLES OF SUBSTRATE AND TETRAHYDROBIOPTERIN IN OXYGEN ACTIVATION

Mammalian NO synthases catalyze the monooxygenation of L-arginine (L-Arg) to N-hydroxyarginine (NOHA) and the subsequent monooxygenation of this to NO and citrulline. Both steps proceed via formation of an oxyferrous heme complex and may ultimately lead to a ferrous NO complex, from which NO must be released. Electrochemical reduction of NO-bound neuronal nitricoxide synthase (nNOS) oxygenase domain was used to form the ferrous heme NO complex, which was found to be stable only in the presence of low NO concentrations, due to catalytic degradation of NO at the nNOS heme site. The reduction potential for the heme-NO complex was approximately -140 mV, which shifted to 0 mV in the presence of either L-Arg or NOHA. This indicates that the complex is stabilized by 14 kJ mol(-1) in the presence of substrate, consistent with a strong H-bonding interaction between NO and the guanidino group. Neither substrate influenced the reduction potential of the ferrous heme CO complex, however. Both L-Arg and NOHA appear to interact with bound NO in a similar way, indicating that both bind as guanidinium ions. The dissociation constant for NO bound to ferrous heme in the presence of l-Arg was determined electrochemically to be 0.17 nM, and the rate of dissociation was estimated to be 10(-4) s(-1), which is much slower than the rate of catalysis. Stopped-flow kinetic analysis of oxyferrous formation and decay showed that both l-Arg and NOHA also stabilize the ferrous heme dioxy complex, resulting in a 100-fold decrease in its rate of decay. Electron transfer from the active-site cofactor tetrahydrobiopterin (H4B) has been proposed to trigger the monoxygenation process. Consistent with this, substitution by the analogue/inhibitor 4-amino-H4B stabilized the oxyferrous complex by a further two orders of magnitude. H4B is required, therefore, to break down both the oxyferrousand ferrous nitrosyl complexes of nNOS during catalysis. The energetics of these processes necessitates an electron donor/acceptor operating within a specific reduction potential range, defining the role of H4B.

Crystal structures of ferrous and ferrous–NO forms of verdoheme in a complex with human heme oxygenase-1: catalytic implications for heme cleavage

Heme oxygenase oxidatively degrades heme to biliverdin resulting in the release of iron and CO through a process in which the heme participates both as a cofactor and substrate. One of the least understood steps in the heme degradation pathway is the conversion of verdoheme to biliverdin. In order to obtain a better understanding of this step we report the crystal structures of ferrous–verdoheme and, as a mimic for the oxy–verdoheme complex, ferrous–NO verdoheme in a complex with human HO-1 at 2.20 and 2.10 Å, respectively. In both structures the verdoheme occupies the same binding location as heme in heme–HO-1, but rather than being ruffled verdoheme in both sets of structures is flat. Both structures are similar to their heme counterparts except for the distal helix and heme pocket solvent structure. In the ferrous–verdoheme structure the distal helix moves closer to the verdoheme, thus tightening the active site. NO binds to verdoheme in a similar bent conformation to that found in heme–HO-1. The bend angle in the verodoheme–NO structure places the terminal NO oxygen 1 Å closer to the α- meso oxygen of verdoheme compared to the α- meso carbon on the heme–NO structure. A network of water molecules, which provide the required protons to activate the iron–oxy complex of heme–HO-1, is absent in both ferrous–verdoheme and the verdoheme–NO structure.

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 p K a values of the coordinated H 2O 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 H 2O or NO and the ionic substituents of the porphyrins.

A novel microperoxidase activity: methyl viologen-linked nitrite reducing activity of microperoxidase

To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO 2 − to NO by donating one electron, the NO was completely reduced to NH 4 + under anaerobic condition via ferrous–NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage. The mp will be able to use as a substitute for nitrite reductase.

Crystal Structures of the Ferric, Ferrous, and Ferrous–NO Forms of the Asp140Ala Mutant of Human Heme Oxygenase-1: Catalytic Implications

Site-directed mutagenesis studies have shown that Asp140 in both human and rat heme oxygenase-1 is critical for enzyme activity. Here, we report the D140A mutant crystal structure in the Fe(III) and Fe(II) redox states as well as the Fe(II)–NO complex as a model for the Fe(II)–oxy complex. These structures are compared to the corresponding wild-type structures. The mutant and wild-type structures are very similar, except for the distal heme pocket solvent structure. In the Fe(III) D140A mutant one water molecule takes the place of the missing Asp140 carboxylate side-chain and a second water molecule, novel to the mutant, binds in the distal pocket. Upon reduction to the Fe(II) state, the distal helix running along one face of the heme moves closer to the heme in both the wild-type and mutant structures thus tightening the active site. NO binds to both the wild-type and mutant in a bent conformation that orients the NO O atom toward the α- meso heme carbon atom. A network of water molecules provides a H-bonded network to the NO ligand, suggesting a possible proton shuttle pathway required to activate dioxygen for catalysis. In the wild-type structure, Asp140 exhibits two conformations, suggesting a dynamic role for Asp140 in shuttling protons from bulk solvent via the water network to the iron-linked oxy complex. On the basis of these structures, we consider why the D140A mutant is inactive as a heme oxygenase but active as a peroxidase.

Proton and electron pathways in the bacterial nitric oxide reductase.

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.

Complex Formation of Cytochrome P450cam with Putidaredoxin: EVIDENCE FOR PROTEIN-SPECIFIC INTERACTIONS INVOLVING THE PROXIMAL THIOLATE LIGAND

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.

Cytochrome P450nor: A nitric oxide reductase?Structure, spectra, and mechanism

The present study characterizes key proposed intermediates and reaction pathway steps in the enzymatic cycle of cytochrome P450nor, a nitric oxide reductase. This unusual P450, with reductase rather than monoxygenase function, catalyzes the conversion of •NO to N2O, an important detoxification step in the global nitrogen cycle. Density functional theoretical (DFT) and semiempirical INDO/ROHF/CI quantum chemical characterization of the putative species in the enzymatic cycle have been made and direct comparisons made with published experimental spectral and structural data. The computed INDO/ROHF-CIS spectra of the NO bound heme species indicate a previously reported experimental transient electronic spectrum to be due the NO bound reduced ferrous heme species. The identity of the active heme species in the P450nor mechanism is unknown and may be either a nitrogen (FeN) N-ferryl analogue of the oxyferryl/compound I P450 species or the NO bound reduced ferrous heme species. Formation of products from either requires the addition of two protons and a second •NO to the NO bound reduced ferrous heme form to form N2O, water, and resting state heme products. The mechanism of proton transfer to the NO bound reduced ferrous NO species is assessed and contrasted with that discovered for the reduced oxyferrous heme species of cytochrome P450 monoxygenases. DFT reaction pathways studies are reported for the addition of •NO to the N-ferryl species and indicate it to be a competent active species forming N2O via an exothermic, barrierless process. © 2002 John Wiley & Sons, Inc. Int J Quantum Chem, 2002