Azide-inhibited bacterial heme oxygenases exhibit an S = 3/2 (dxz,dyz)3(dxy)1(dz2)1 spin state: mechanistic implications for heme oxidation

The azide complexes of heme oxygenase from Pseudomonas aeruginosa (pa-HO) and Neisseriae meningitidis (nm-HO) have been studied with the aid of (1)H and (13)C NMR spectroscopy. These complexes have been shown to exist as an equilibrium mixture of two populations, one exhibiting an S = (1)/(2), (d(xy))(2)(d(xz), d(yz))(3) electron configuration and planar heme and a second with a novel S = (3)/(2), (d(xz), d(yz))(3)(d(xy))(1)(d(z)(2))(1) spin state and nonplanar heme. At physiologically relevant temperatures, the equilibrium shifts in the direction of the population exhibiting the latter electron configuration and nonplanar heme, whereas at temperatures approaching the freezing point of water, the equilibrium shifts in the direction of the population with the former electronic structure and planar heme. These findings indicate that the microenvironment of the distal pocket in heme oxygenase is unique among heme-containing proteins in that it lowers the sigma-donating (field strength) ability of the distal ligand and, therefore, promotes the attainment of heme electronic structures thus far only observed in heme oxygenase. When the field strength of the distal ligand is slightly lower than that of azide, such as OH(-) (J. Am. Chem. Soc. 2003, 125, 11842), the corresponding complex exists as a mixture of populations with nonplanar hemes and electronic structures that place significant spin density at the meso positions. The ease with which these unusual heme electronic structures are attained by heme oxygenase is likely related to activation of meso carbon reactivity which, in turn, facilitates hydroxylation of a meso carbon by the obligatory ferric hydroperoxide intermediate.     

The effects of axial ligands on electron distribution and spin states in iron complexes of octaethyloxophlorin, intermediates in heme degradation

The results presented here show that the nature of the axial ligand can alter the distribution of electrons between the metal and the porphyrin in complexes where there is an oxygen atom replacing one of the meso protons. The complexes (1-MeIm)(2)Fe(III)(OEPO) and (2,6-xylylNC)(2)Fe(II)(OEPO(*)) (where OEPO is the trianionic octaethyloxophlorin ligand and OEPO(*) is the dianionic octaethyloxophlorin radical) were prepared by addition of an excess of the appropriate axial ligand to a slurry of [Fe(III)(OEPO)](2) in chloroform under anaerobic conditions. The magnetic moment of (2,6-xylylNC)(2)Fe(II)(OEPO(*)) is temperature invariant and consistent with a simple S = (1)/(2) ground state. This complex with an EPR resonance at g = 2.004 may be considered as a model for the free-radical like EPR signal seen when the meso-hydroxylated heme/heme oxygenase complex is treated with carbon monoxide. In contrast, the magnetic moment of (1-MeIm)(2)Fe(III)(OEPO) drops with temperature and indicates a spin-state change from an S = (5)/(2) or an admixed S = (3)/(2),(5)/(2) state at high temperatures (near room temperature) to an S = (1)/(2) state at temperatures below 100 K. X-ray diffraction studies show that each complex crystallizes in centrosymmetric form with the expected six-coordinate geometry. The structure of (1-MeIm)(2)Fe(III)(OEPO) has been determined at 90, 129, and 296 K and shows a gradual and selective lengthening of the Fe-N(axial bond). This behavior is consistent with population of a higher spin state at elevated temperatures.     

Coupled oxidation vs heme oxygenation: insights from axial ligand mutants of mitochondrial cytochrome b5

Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b(5) (OM cyt b(5)), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O(2). The reaction proceeds via the formation of an oxyferrous complex (Fe(II)(-)O(2)) that is reduced by hydrazine to a ferric hydroperoxide (Fe(III)(-)OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (Fe(III)(-)OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b(5), to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of Fe(III)(-)H63V with H(2)O(2) leads to the nonspecific degradation of heme, the incubation of Fe(II)(-)H63V with H(2)O(2) results in the formation of meso-hydroxyheme, which upon exposure to O(2) is rapidly converted to verdoheme. These findings revealed that although meso-hydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O(2) is reduced to noncoordinated H(2)O(2), which reacts with Fe(II)-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O(2) molecule (Fe(II)(-)O(2)) is reduced to a coordinated peroxide molecule (Fe(III)(-)OOH), which oxidizes heme to meso-hydroxyheme.     

Quantum mechanical/molecular mechanical study of mechanisms of heme degradation by the enzyme heme oxygenase: the strategic function of the water cluster

Heme degradation by heme oxygenase (HO) enzymes is important in maintaining iron homeostasis and prevention of oxidative stress, etc. In response to mechanistic uncertainties, we performed quantum mechanical/molecular mechanical investigations of the heme hydroxylation by HO, in the native route and with the oxygen surrogate donor H2O2. It is demonstrated that H2O2 cannot be deprotonated to yield Fe(III)OOH, and hence the surrogate reaction starts from the FeHOOH complex. The calculations show that, when starting from either Fe(III)OOH or Fe(III)HOOH, the fully concerted mechanism involving O-O bond breakage and O-C(meso) bond formation is highly disfavored. The low-energy mechanism involves a nonsynchronous, effectively concerted pathway, in which the active species undergoes first O-O bond homolysis followed by a barrier-free (small with Fe(III)HOOH) hydroxyl radical attack on the meso position of the porphyrin. During the reaction of Fe(III)HOOH, formation of the Por+*FeIV=O species, compound I, competes with heme hydroxylation, thereby reducing the efficiency of the surrogate route. All these conclusions are in accord with experimental findings (Chu, G. C.; Katakura, K.; Zhang, X.; Yoshida, T.; Ikeda-Saito, M. J. Biol. Chem. 1999, 274, 21319). The study highlights the role of the water cluster in the distal pocket in creating "function" for the enzyme; this cluster affects the O-O cleavage and the O-Cmeso formation, but more so it is responsible for the orientation of the hydroxyl radical and for the observed alpha-meso regioselectivity of hydroxylation (Ortiz de Montellano, P. R. Acc. Chem. Res. 1998, 31, 543). Differences/similarities with P450 and HRP are discussed.     

Spin crossover in a tetranuclear Cr(III)-Fe(III)(3) complex

A novel heteronuclear exchange-coupled complex [Cr(III)[(CN)Fe(III)((5)L)](3)(CN)(3)] containing a pentadentate blocking ligand (5)L was synthesized. The X-ray structure shows that a meridional isomer applies with inequivalent Fe(III) centers. The complex exhibits a thermally induced spin crossover along with the exchange coupling. M?ssbauer spectra indicate a spin transition between S = (1)/(2) and S = (5)/(2) states although a considerable amount of Fe(III) centers stays high-spin at T = 6 K. The magnetization, the magnetic susceptibility, and the M?ssbauer data were fitted in one run with a spin crossover model taking into account exchange interactions among all metal centers.     

The hydrogen-bonding network in heme oxygenase also functions as a modulator of enzyme dynamics: chaotic motions upon disrupting the H-bond network in heme oxygenase from Pseudomonas aeruginosa

Relaxation compensated Carr-Purcell-Meiboom-Gill (rc-CPMG) NMR experiments have been used to investigate micros-ms motions in heme oxygenase from Pseudomonas aeruginosa (pa-HO) in its ferric state, inhibited by CN- (pa-HO-CN) and N3- (pa-HO-N3), and in its ferrous state, inhibited by CO (pa-HO-CO). Comparative analysis of the data from the three forms indicates that the nature of the coordinated distal ligand affects the micros-ms conformational freedom of the polypeptide in regions of the enzyme far removed from the heme iron and distal ligand. Interpretation of the dynamical information in the context of the crystal structure of resting state pa-HO shows that residues involved in the network of structural hydrogen-bonded waters characteristic of HOs undergo micros-ms motions in pa-HO-CN, which was studied as a model of the highly paramagnetic S = 5/2 resting state form. In comparison, similar motions are suppressed in the pa-HO-CO and pa-HO-N3 complexes, which were studied as mimics of the obligatory oxyferrous and ferric hydroperoxide intermediates, respectively, in the catalytic cycle of heme degradation. These findings suggest that in addition to proton delivery to the nascent Fe(III)-OO(-) intermediate during catalysis, the hydrogen-bonding network serves two additional roles: (i) propagate the electronic state (reactive state) in each of the distinct steps of the catalytic cycle to key but remote sections of the polypeptide via small rearrangements in the network of hydrogen bonds and (ii) modulate the conformational freedom of the enzyme, thus allowing it to adapt to the demanding changes in axial coordination state and substrate transformations that take place during the catalytic cycle. This idea was probed by disrupting the hydrogen-bonding network in pa-HO by replacing R80 with L. NMR spectroscopic studies conducted with R80L-pa-HO-N3 and R80L-pa-HO-CO revealed that the mutant exhibits nearly global conformational disorder, which is absent in the equivalent complexes of the wild type enzyme. The "chaotic" disorder in the R80L mutant is likely related to its significantly lower efficiency to hydroxylate heme in the presence of H2O2, relative to the wild type enzyme.     

Ligand-field dependence of the excited state dynamics of Hangman bisporphyrin dyad complexes

A new Hangman porphyrin architecture has been developed to interrogate the ligand-field dependence of photoinduced PCET versus excitation energy transfer and intersystem crossing in PZn(II)-PFe(III)-OH dyads (P = porphyrin). In this design, a hanging carboxylic acid group establishes a hydrogen-bonding network to anchor the weak-field OH- ligand in the distal site of the PFe(III)-OH acceptor, whereas the proximal site is left available to accept strong-field imidazole ligands. Thus, controlling the tertiary coordination environment gives access to the first synthetic example of a porphyrin dyad with a biologically relevant weak-field/strong-field configuration of axial ligands at the heme. Transient absorption spectroscopy has been employed to probe the fate of the initial PZn(II)-based S1 excited state, revealing rapid S1 quenching for all dyads in the presence and absence of strong-field imidazole ligands (tau = 6-50 ps). The absence of a (P*+)Zn(II) signal that would complement photoinduced PCET at the PFe(III)-OH subunit (i.e., PFe(III)-OH --> PFe(II)-OH2) shows that excitation energy transfer and intersystem crossing channels dominate the quenching, regardless of whether proximal strong field ligands are present. Moreover, this photophysical assignment is independent of the solvent dielectric constant and whether a phenylene or biphenylene spacer is used to span the two porphyrin subunits. Electronic structure calculations suggest that the structural reorganization attendant to reductive PCET at the high-spin Fe(III)-OH center imposes a severe kinetic cost that can only be alleviated by inducing a low-spin electronic configuration with two strong-field axial ligands.     

Spin Crossover in Nitrito‐Myoglobin as Revealed by Resonance Raman Spectroscopy

The myoglobin (Mb) heme Fe‐O‐N=O and heme Fe‐O‐N=O/2‐nitrovinyl species have been characterized by resonance Raman spectroscopy. In the heme Fe‐O‐N=O species, the bound nitrite ligand is removed by solvent exchange, thus reforming metmyoglobin (metMb). The high‐spin heme Fe‐O‐N=O unit is converted into a low‐spin heme Fe‐O‐N=O/2‐nitrovinyl species that can be reversibly switched between a low‐ and a high‐spin state without removing the bound nitrite ligand, as observed in the case of the heme Fe‐O‐N=O species. This spin‐state change is likely to be accompanied by a general structural rearrangement in the protein‐binding pocket. This example is the first of a globin protein that can reversibly change its metal spin state through an internal perturbation. These findings provide a basis for understanding the structure–function relationship of the spin cross found in other metalloenzymes and Fe^III–porphyrin complexes.     

S = (3)/(2) <= => S = (1)/(2) spin crossover behavior in five-coordinate halido- and pseudohalido-bis(o-iminobenzosemiquinonato)iron(III) complexes

Five-coordinate halido- and pseudohalido-bis(o-iminobenzosemiquinonato)iron(III) complexes [Fe(III)X(L(ISQ))(2)] (X = Cl(-) (1), Br(-) (2a, 2b), I(-) (3), N(3)(-) (4), and NCS(-) (5)) have been synthesized where (L(ISQ))(1)(*)(-) represents the pi radical anion N-phenyl-o-imino(4,6-di-tert-butyl)benzosemiquinonate(1-). The molecular structures of the two polymorphs 2a and 2b have been determined at 100, 220, and 295 K, respectively, by single crystal X-ray crystallography. Variable temperature magnetic susceptibility data reveal the following electronic ground states, S(t): For 1, it is (3)/(2). Polymorph 2a contains a 1:1 mixture of (3)/(2) and (1)/(2) forms in the range 4.2 to approximately 150 K; above 150 K the latter form undergoes a spin crossover (1)/(2) --> (3)/(2). Polymorph 2b contains only the S(t) = (3)/(2) form (4-300 K). Complex 3 contains the S(t) = (1)/(2) form in the range 4-130 K, but above 130 K, a spin crossover to the (3)/(2) form is observed which is confirmed by three crystal structure determinations at 100, 220, and 295 K. Complex 4 possesses an S(t) = (1)/(2) ground state at 80 K and undergoes a spin crossover at higher temperatures. Complex 5 has a temperature-independent S(t) = (3)/(2) ground state. All crystal structures of 1, 2a, 2b, 3, 4, and 5, regardless at which temperature the data sets have been measured, show that two o-iminobenzosemiquinonate(1-) pi radical anions are N,O-coordinated in all of these neutral iron complexes. The Fe-N and Fe-O bond distances are longer in the S(t) = (3)/(2) and shorter in the S(t) = (1)/(2) forms. The S(t) = (3)/(2) ground state is attained via intramolecular antiferromagnetic coupling between a high spin ferric ion (S(Fe) = (5)/(2)) and two ligand pi radicals whereas the S(t) = (1)/(2) form is generated from exchange coupling between an intermediate spin ferric ion (S(Fe) = (3)/(2)) and two ligand radicals.     

Ibuprofen induces an allosteric conformational transition in the heme complex of human serum albumin with significant effects on heme ligation

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.     

Is the corrolate macrocycle innocent or noninnocent? Magnetic susceptibility,Mössbauer, 1H NMR,and DFT investigations of chloro- and phenyliron corrolates

In an attempt to determine the electron configuration of (anion)iron corrolates, i.e., whether they are S = 1 Fe(IV)-corrolate(3-) or S = 3/2 Fe(III)-corrolate(2-*), with antiferromagnetic coupling between the iron and macrocycle electrons to yield overall S = 1, two axial ligand complexes of an iron octaalkylcorrolate have been studied by temperature-dependent magnetic susceptibility, magnetic M?ssbauer, and 1H NMR spectroscopy, and the results have been compared to those determined on the basis of spin-unrestricted DFT calculations. Magnetic susceptibility measurements indicate the presence of a noninnocent macrocycle (corrolate (2-*)) for the chloroiron corrolate, with strong antiferromagnetic coupling to the S = 3/2 Fe(III) center, while those for the phenyliron corrolate are not conclusive as to the electron configuration. Temperature- and field-dependent M?ssbauer spectroscopic investigations of these two complexes yielded spectra that could be simulated with either electron configuration, except that the isomer shift of the phenyl-iron complex is -0.10 mm/s while that of the chloroiron complex is +0.21 mm/s, suggesting that the iron in the former is Fe(IV) while in the latter it is Fe(III). 1H NMR spectroscopic studies of both axial ligand complexes show large negative spin density at the meso carbons, with those of the chloroiron complex (Cai, S.; Walker, F. A.; Licoccia, S. Inorg. Chem. 2000, 39, 3466) being roughly four times larger than those of the phenyliron complex. The temperature dependence of the proton chemical shifts of the phenyliron complex is strictly linear. DFT calculations are consistent with the chloroiron complex being formulated as S1 = 3/2 Fe(III)-corrolate (2-*) S2 = 1/2, with negative spin density at all nitrogens and meso carbons, and a net spin density of -0.79 on the corrolate ring and positive spin density (+0.17) on the chloride ion and +2.58 on the iron. In contrast, the phenyliron complex is best formulated as S = 1 Fe(IV)-corrolate (3-), but again with negative spin density at all nitrogens and meso carbons of the macrocycle, yet with the net spin density on the corrolate ring being virtually zero; the phenyl carbanion carbon has relatively large negative spin density of -0.15 and the iron +2.05. On the basis of all of the results, we conclude that in both the chloroiron and phenyliron complexes the corrolate ring is noninnocent, in the chloroiron complex to a much larger extent than in the phenyliron complex.     

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.     

Static normal coordinate deformations of the heme group in mutants of ferrocytochrome c from Saccharomyces cerevisiae probed by resonance Raman spectroscopy

The function of heme proteins is, to a significant extent, influenced by the ligand field probed by the heme iron, which itself can be affected by deformations of the heme macrocycle. The exploration of this field is difficult because the heme structure obtained from X-ray crystallography is not resolved enough to unambiguously identify structural changes on the scale of 10(-2) A. However, asymmetric deformations in this order of magnitude affect the depolarization ratio of the resonance Raman lines assignable to normal vibrations of the heme group. We have measured the dispersion of the depolarization ratios of four structure sensitive Raman bands (i.e., nu4, nu11, nu21, and nu28) in yeast iso-1-ferrocytochrome c and its mutants N52V, Y67F, and N52VY67F with B- and Q-band excitation. The DPR dispersion of all bands indicates the presence of asymmetric in-plane and out-of-plane deformations. The replacement of the polar tyrosine residue at position 67 by phenylalanine significantly increases the triclinic B2g deformation, which involves a distortion of the pyrrole symmetry. We relate this deformation to changes of the electronic structure of pyrrole A, which modulates the interaction between its propionate substituents and the protein environment. This specific heme deformation is eliminated in the double mutant N52VY67F. The additional substitution of N52 by valine induces a tetragonal B1g deformation which involves asymmetric changes of the Fe-N distances and increases the rhombicity of the ligand field probed by the heme iron. This heme deformation might be caused by the elimination of the water-protein hydrogen-bonding network in the heme cavity. The single mutation N52V does not significantly perturb the heme symmetry, but a small B1g deformation is consistent with our data and the heme structure obtained from a 1 ns molecular dynamics simulation of the protein.     

Differential sensing of protein influences by NO and CO vibrations in heme adducts

Heme proteins bind the gaseous ligands XO (X = C, N, O) via backbonding from Fe d(pi) electrons. Backbonding is modulated by distal interactions of the bound ligand with the surrounding protein and by variations in the strength of the trans proximal ligand. Vibrational modes associated with FeX and XO bond stretching coordinates report on these interactions, but the interpretive framework developed for CO adducts, involving anticorrelations of nuFeC and nuCO, has seemed not to apply to NO adducts. We have now obtained an excellent anticorrelation of nuFeN and nuNO, via resonance Raman spectroscopy on (N-methylimidazole)Fe(II)TPP-Y(NO), where TPP-Y is tetraphenylporphine with electron-donating or -withdrawing substituents, Y, that modulate the backbonding; the problem of laser-induced dissociation of the axial base was circumvented by using frozen solutions. New data are also reported for CO adducts. The anticorrelations are supported by DFT calculations of structures and spectra. When protein data are examined, the NO adducts show large deviations from the modeled anticorrelation when there are distal H-bonds or positive charges. These deviations are proposed to result from closing of the FeNO angle due to a shift in the valence isomer equilibrium toward the Fe(III)(NO-) form, an effect that is absent in CO adducts. The differing vibrational patterns of CO and NO adducts provide complementary information with respect to protein interactions, which may help to elucidate the mechanisms of ligand discrimination and signaling in heme sensor proteins.     

Origin of the red shifts in the optical absorption bands of nonplanar tetraalkylporphyrins

The view that the large red shifts seen in the UV-visible absorption bands of peripherally crowded nonplanar porphyrins are the result of nonplanar deformations of the macrocycle has recently been challenged by the suggestion that the red shifts arise from substituent-induced changes in the macrocycle bond lengths and bond angles, termed in-plane nuclear reorganization (IPNR). We have analyzed the contributions to the UV-visible band shifts in a series of nickel or zinc meso-tetraalkylporphyrins to establish the origins of the red shifts in these ruffled porphyrins. Structures were obtained using a molecular mechanics force field optimized for porphyrins, and the nonplanar deformations were quantified by using normal-coordinate structural decomposition (NSD). Transition energies were calculated by the INDO/S semiempirical method. These computational studies demonstrate conclusively that the large Soret band red shifts ( approximately 40 nm) seen for very nonplanar meso-tetra(tert-butyl)porphyrin compared to meso-tetra(methyl)porphyrin are primarily the result of nonplanar deformations and not IPNR. Strikingly, nonplanar deformations along the high-frequency 2B(1u) and 3B(1u) normal coordinates of the macrocycle are shown to contribute significantly to the observed red shifts, even though these deformations are an order of magnitude smaller than the observed ruffling (1B(1u)) deformation. Other structural and electronic influences on the UV-visible band shifts are discussed and problems with the recent studies are examined (e.g., the systematic underestimation of the 2B(1u) and 3B(1u) modes in artificially constrained porphyrin structures that leads to a mistaken attribution of the red shift to IPNR). The effect of nonplanar deformations on the UV-visible absorption bands is then probed experimentally with a series of novel bridled nickel chiroporphyrins. In these compounds, the substituent effect is essentially invariant and the amount of nonplanar deformation decreases as the length of the straps connecting adjacent meso-cyclopropyl substituents decreases (the opposite of the effect observed for conventional strapped porphyrins). Several spectroscopic markers for nonplanarity (UV-visible bands, resonance Raman lines, and (1)H NMR resonances) are found to correlate with time-averaged deformations obtained from an NSD analysis of molecular dynamics snapshot structures. These results suggest that UV-visible band shifts of tetrapyrroles in proteins are potentially useful indicators of changes in nonplanarity provided other structural and electronic factors can be eliminated.     

The ferrous verdoheme-heme oxygenase complex is six-coordinate and low-spin

A biosynthetic and enzymatic method was developed for the preparation of 13C-labeled verdoheme, which permits the 13C NMR spectroscopic characterization of this elusive intermediate in the heme oxidation path catalyzed by the enzyme heme oxygenase. The 13C NMR data indicate that the ferrous verdoheme complex of Neisseria meningitides heme oxygenase is hexacoordinate and diamagnetic, with a proximal histidine and likely a distal hydroxide as axial ligands. The coordination number and spin state of the ferrous verdoheme-heme oxygenase complex is in stark contrast to the pentacoordinate and paramagnetic nature of the heme-heme oxygenase complex and heme centers in general.     

Xanthene-modified and hangman iron corroles

Iron corroles modified with a xanthene scaffold are delivered from easily available starting materials in abbreviated reaction times. These new iron corroles have been spectroscopically examined with particular emphasis on defining the oxidation state of the metal center. Investigation of their electronic structure using (57)Fe Mo?ssbauer spectroscopy in conjunction with density functional theory (DFT) calculations reveals the non-innocence of the corrole ligand. Although these iron corroles contain a formal Fe(IV) center, the deprotonated corrole macrocycle ligand is one electron oxidized. The electronic ground state of these complexes is best described as an intermediate spin S = 3/2 Fe(III) site strongly antiferromagnetically coupled to the S = 1/2 of the monoradical dianion corrole [Fe(III)Cl-corrole(+?)]. We show here that iron corroles as well as xanthene-modified and hangman xanthene iron corroles are redox active and catalyze the disproportionation of hydrogen peroxide via the catalase reaction, and that this activity scales with the oxidation potential. The meso position of corrole macrocycle is susceptible toward nucleophilic attack during catalase turnover. The reactivity of peroxide within the hangman cleft reported here adds to the emerging theme that corroles are good at catalyzing two-electron activation of the oxygen-oxygen bond in a variety of substrates.     

Genetic engineering of the heme pocket in human serum albumin: modulation of O2 binding of iron protoporphyrin IX by variation of distal amino acids

Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport.     

A single-site mutation (F429H) converts the enzyme CYP 2B4 into a heme oxygenase: a QM/MM study

The intriguing deactivation of the cytochrome P450 (CYP) 2B4 enzyme induced by mutation of a single residue, Phe429 to His, is explored by quantum mechanical/molecular mechanical calculations of the O-OH bond activation of the (Fe(3+)OOH)(-) intermediate. It is found that the F429H mutant of CYP 2B4 undergoes homolytic instead of heterolytic O-OH bond cleavage. Thus, the mutant acquires the following characteristics of a heme oxygenase enzyme: (a) donation by His429 of an additional NH---S H-bond to the cysteine ligand combined with the presence of the substrate retards the heterolytic cleavage and gives rise to homolytic O-OH cleavage, and (b) the Thr302/water cluster orients nascent OH(?) and ensures efficient meso hydroxylation.     

N,N-coordinated pi radical anions of S-methyl-1-phenyl-isothiosemicarbazide in two five-coordinate ferric complexes [Fe III(L Me*)(2)X] (X = CH3S-, Cl-)

The reaction between [Fe(III)(dmf)(6)](ClO(4))(3) and the ligand S-methyl-1-phenyl-isothiosemicarbazide, H(2)[L(Me)], and triethylamine (1:3:6) in methanol under an argon blanketing atmosphere at elevated temperatures (reflux) yields a purple solution from which upon cooling to 20 degrees C dark green crystals of [Fe(III)(L(Me)(*))(2)(SCH(3))] (1) were obtained in 15% yield. From a similar reaction mixture using FeCl(3) as starting material in the solvent acetone under anaerobic conditions at -80 degrees C, dark green crystals of [Fe(III)(L(Me)(*))(2)Cl] (2) were obtained in 21% yield. The structures of complexes 1 and 2 have been determined by single-crystal X-ray crystallography at 100 K. Both complexes are five-coordinate square base pyramidal ferric species containing two N,N-coordinated, monoanionic pi radicals, (L(Me)(*))(1)(-), of the parent S-methyl-1-phenyl-isothiosemicarbazide(2-) dianion in the basal positions whereas the axial position is occupied by methylthiolate in 1 and chloride in 2, respectively. The electronic structure of both species has been elucidated by their electronic spectra, magnetic properties, and X-band EPR and M?ssbauer spectra. Both possess an S(t) = (1)/(2) ground state which is attained via an antiferromagnetic coupling between the spins of an intermediate spin ferric ion (S(Fe) = (3)/(2)) and two ligand pi radical anions (S(rad) = (1)/(2)).