Probing Bis-FeIV MauG: Isolation of Highly Reactive Radical Intermediates.

MauG is a diheme enzyme that utilizes two covalently bound c-type heme centers. We report here step-wise oxidations of a synthetic analogue of MauG in which two heme centers are bridged covalently through a flexible linker containing a pyrrole moiety. One- and two-electron oxidations produce monocation radical and dication diradical intermediates, respectively, which, being highly reactive, undergo spontaneous intramolecular rearrangement involving the pyrrole bridge itself to form indolizinium-fused chlorin-porphyrin and spiro-porphyrinato heterodimers. Unlike in MauG, where the two oxidizing equivalents produce the bis-FeIV redox state, the synthetic analogue of the same, however, stabilizes two ferric hemes, each coupled with a porphyrin π-cation radical. The present study highlights the possible role played by the bridge in the electronic communication.

Design of Heteronuclear Metalloenzymes.

Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedures on how to choose the protein scaffold, design a heterobinuclear metal center in the protein scaffold computationally, incorporate metal ions into the protein, and characterize the resulting metalloproteins, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.

Anisotropic iron motion in nitrosyl iron porphyrinates: natural and synthetic hemes.

The vibrational spectra of two five-coordinate nitrosyl iron porphyrinates, [Fe(OEP)(NO)] (OEP = dianion of 2,3,7,8,12,13,17,18-octaethylporphyrin) and [Fe(DPIX)(NO)] (DPIX = deuteroporphyrin IX), have been studied by oriented single-crystal nuclear resonance vibrational spectroscopy. Single crystals (both are in the triclinic crystal system) were oriented to give vibrational spectra perpendicular to the porphyrin plane. Additionally, two orthogonal in-plane measurements that were also either perpendicular or parallel to the projection of the FeNO plane onto the porphyrin plane yield the complete set of vibrations with iron motion. In addition to cleanly enabling the assignment of the FeNO bending and stretching modes, the measurements reveal that the two in-plane spectra from the parallel and perpendicular in-plane directions for both compounds have substantial differences. The assignment of these in-plane vibrations were aided by density functional theory predictions. The differences in the two in-plane directions result from the strongly bonded axial NO ligand. The direction of the in-plane iron motion is thus found to be largely parallel and perpendicular to the projection of the FeNO plane on the porphyrin plane. These axial ligand effects on the in-plane iron motion are related to the strength of the axial ligand-to-iron bond.

Electronic structure and biologically relevant reactivity of low-spin {FeNO}8 porphyrin model complexes: new insight from a bis-picket fence porphyrin.

Because of HNO's 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.

Synthesis and antimalarial activity of urenyl Bis-chalcone in vitro and in vivo.

Some novel derivatives of Bis-chalcone were synthesized and characterized by their physical and spectral data. All the synthesized compounds were subsequently screened for in vitro globin hydrolysis, ß-hematin formation, and murine Plasmodium berghei, using chloroquine as the reference drug. Most of the synthesized compounds exhibited mild to moderate susceptibilities toward the parasite in comparison with the standard. The most active antimalarial compound was 1,1-Bis-[(3',4'-N-(urenylphenyl)-3-(3″,4″,5″-trimethoxyphenyl)]-2-propen-1-one 5, with a percentage of inhibition of heme polymerization of 87.05 ± 0.77, and this compound increased the survival time after infection, reduce the parasitemia and delay the progression of malaria.

A new, simple method for the production of meat-curing pigment under optimised conditions using response surface methodology.

The production of cured meat pigment using nitrite and ascorbate in acidic conditions was evaluated. HCl, ascorbate and nitrite concentrations were optimised at three levels using the response surface method (RSM). The effects of process variables on the nitrosoheme yield, the wavelength of maximum absorbance (λ(max)), and L*, a* and b* values were evaluated. The response surface equations indicate that variables exerted a significant effect on all dependent factors. The optimum combinations for the reaction were HCl=-0.8, ascorbate=0.46 and nitrite=1.00 as coded values for conversion of 1mM hemin to nitrosoheme, by which a pigment yield of 100%, which was similar to the predicted value of 99.5%, was obtained. Likewise, the other parameters were not significantly different from predicted values as the λ(max), L*, a* and b* values were 558 nm, 47.03, 45.17 and 17.20, respectively. The structure of the pigment was identified using FTIR and ESI/MS.

Molecular hijacking of siroheme for the synthesis of heme and d1 heme.

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

Proton-coupled electron transfer reactions at a heme-propionate in an iron-protoporphyrin-IX model compound.

A heme model system has been developed in which the heme-propionate is the only proton donating/accepting site, using protoporphyrin IX-monomethyl esters (PPIX(MME)) and N-methylimidazole (MeIm). Proton-coupled electron transfer (PCET) reactions of these model compounds have been examined in acetonitrile solvent. (PPIX(MME))Fe(III)(MeIm)(2)-propionate (Fe(III)~CO(2)) is readily reduced by the ascorbate derivative 5,6-isopropylidine ascorbate to give (PPIX(MME))Fe(II)(MeIm)(2)-propionic acid (Fe(II)~CO(2)H). An excess of the hydroxylamine TEMPOH or of hydroquinone similarly reduces Fe(III)~CO(2), and TEMPO and benzoquinone oxidize Fe(II)~CO(2)H to return to Fe(III)~CO(2). The measured equilibrium constants, and the determined pK(a) and E(1/2) values, indicate that Fe(II)~CO(2)H has an effective bond dissociation free energy (BDFE) of 67.8 ± 0.6 kcal mol(-1). In these PPIX models, electron transfer occurs at the iron center and proton transfer occurs at the remote heme propionate. According to thermochemical and other arguments, the TEMPOH reaction occurs by concerted proton-electron transfer (CPET), and a similar pathway is indicated for the ascorbate derivative. Based on these results, heme propionates should be considered as potential key components of PCET/CPET active sites in heme proteins.

Horseradish peroxidase: modulation of properties by chemical modification of protein and heme.

Horseradish peroxidase (HRP) is one of the most studied enzymes of the plant peroxidase superfamily. HRP is also widely used in different bioanalytical applications and diagnostic kits. The methods of genetic engineering and protein design are now widely used to study the catalytic mechanism and to improve properties of the enzyme. Here we review the results of another approach to HRP modification-through the chemical modification of amino acids or prosthetic group of the enzyme. Computer models of HRPs with modified hemes are in good agreement with the experimental data.

Catalytic reduction of O2 by cytochrome C using a synthetic model of cytochrome C oxidase.

Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the one-electron reductant Cytochrome c (Cytc) being the source of electrons. Recently we reported a functional model of CcO that electrochemically catalyzes the four-electron reduction of O(2) to H(2)O (Collman et al. Science 2007, 315, 1565). The current paper shows that the same functional CcO model catalyzes the four-electron reduction of O(2) using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetics studies indicate that O(2) binding is rate-determining and that O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are relatively fast.

A stable hyponitrite-bridged iron porphyrin complex.

The coupling of two nitric oxide (NO) molecules in heme active sites is an important contributor to the conversion of NO to nitrous oxide (N(2)O) by heme-containing enzymes. Several formulations for the presumed heme-Fe{N(2)O(2)}(n-) intermediates have been proposed previously, however, no crystal structures of heme-Fe{N(2)O(2)}(n-) systems have been reported to date. We report the first isolation and characterization of a stable bimetallic hyponitrite iron porphyrin, [(OEP)Fe](2)(mu-N(2)O(2)), prepared from the reaction of [(OEP)Fe](2)(mu-O) with hyponitrous acid. Density functional theoretical calculations were performed on the model compound [(porphine)Fe](2)(mu-N(2)O(2)) to characterize its electronic structure and properties.

Modulation of metal displacements in a saddle distorted macrocycle: synthesis, structure, and properties of high-spin Fe(III) porphyrins and implications for the hemoproteins.

A rare family of five and six-coordinated high-spin Fe(III) porphyrins incorporating weak axial ligands are synthesized and structurally characterized which demonstrate, for the first time, stepwise metal displacements in a single distorted macrocyclic environment that has generally been seen in many biological systems. The introduction of four nitro groups into the meso-positions of octaethyl porphyrin severely distorts the porphyrin geometry and provides an interesting modulation of the macrocycle properties which enables the facile isolation of "pure" high-spin Fe(III)(tn-OEP)Cl, Fe(III)(tn-OEP)(MeOH)Cl, and Fe(III)(tn-OEP)(H2O)2(+) in excellent yields in a saddle distorted macrocyclic environment that are known to stabilize intermediate spin states. The stepwise out-of-plane displacements of iron are as follows: 0.47 A for Fe(III)(tn-OEP)Cl; 0.09 A for Fe(III)(tn-OEP)(MeOH)Cl, and 0.01 A for Fe(III)(tn-OEP)(H2O)2(+) from the mean plane of the porphyrins. However, in both five and six-coordinated Fe(III) porphyrins, the Fe-Np distances are quite comparable while the porphyrin cores have expanded significantly, virtually to the same extent for the six-coordinate complexes reported here. The large size of the high-spin iron(III) atom in Fe(III)(tn-OEP)(H2O)2(+) is accommodated perfectly with no displacement of the metal. This expansion is accompanied by a significant decrease of the saddle distortion with a clear increase of the ruffling. Furthermore, the Fe atom in Fe(III)(tn-OEP)(MeOH)Cl is not out of plane because of the larger atom size; however, the displacement of the iron depends on both the relative strength of the axial ligands, as well as the nature and extent of the ring deformation. Our characterization demonstrates that increase in ruffling and/or decrease in macrocycle deformation brings the iron atom more into the plane in a distorted macrocyclic environment. Our observations thus suggest that the displacements of iron in proteins are the consequences of nonequivalent axial coordination, as well as protein induced deformations at the heme. The high-spin nature of the complexes reported here is believed to be due to the larger Fe-Np distances which then reduce substantially the interaction between iron d(x2)-y2 and porphyrin a(2u) orbital. The Fe(III)/Fe(II) reduction potential of Fe(III)(tn-OEP)Cl shows a reversible peak at large positive value (0.20 V), and no ring-centered oxidation was observed within the solvent limit (approximately 1.80 V). It is thus easier to reduce Fe(III)(tn-OEP)Cl by almost 700 mV compared to Fe(III)(OEP)Cl while oxidations are very difficult. Furthermore, the addition of 3-Cl-pyridine to Fe(III)(tn-OEP)Cl in air undergoes spontaneous auto reduction to produce the rare air-stable Fe(II)(tn-OEP)(3-Cl-py)2 that shows Fe(II)/Fe(III) oxidation peaks at high positive potential (0.79 V), which is approximately 600 mV more anodic compared to [Fe(II)(tn-OEP)Cl](-). This large anodic shift illustrates the effective removal of metal-centered electron density by the macrocycle when the metal is constrained to reside in the porphyrin plane.

Weak-field anions displace the histidine ligand in a synthetic heme peptide but not in N-acetylmicroperoxidase-8: possible role of heme geometry differences.

We have recently reported that aquo and thioether complexes of the ferric cytochrome c heme peptide N-acetylmicroperoxidase-8 (FeIII-1) exhibit greater low-spin character than do the corresponding complexes of a synthetic, water-soluble, monohistidine-ligated heme peptide (FeIII-2; Cowley, A. B.; Lukat-Rodgers, G. S.; Rodgers, K. R.; Benson, D. R. Biochemistry 2004, 43, 1656-1666). Herein we report results of studies showing that weak-field ligands bearing a full (fluoride, chloride, hydroxide) or partial (phenoxide, thiocyanate) negative charge on the coordinating atom trigger dissociation of the axial His ligand in FeIII-2 but not in FeIII-1. We attribute the greater sensitivity of His ligation in FeIII-1 to weak-field anionic ligands than to weak-field neutral ligands to the following phenomena: (1) anionic ligands pull FeIII further from the mean plane of a porphyrin than do neutral ligands, which will have the effect of straining the His-Fe bond in FeIII-2, and (2) heme in FeIII-2 is likely to undergo a modest doming distortion following anion binding that will render the His-ligated side of the porphyrin concave, thereby increasing porphyrin/ligand steric interactions. We propose that ruffling of the heme in FeIII-1 is an important factor contributing to its ability to resist His dissociation by weak-field anions. First, ruffling should allow His to more closely approach the porphyrin than is possible in FeIII-2, thereby reducing bond strain following anion binding. Second, the ruffling deformation in FeIII-1, which is enforced by the double covalent heme-peptide linkage, will almost certainly prevent significant porphyrin doming.

Coordination of diatomic ligands to heme: simply CO.

The synthesis and molecular structures of three iron(II) porphyrinates with only CO as the axial ligand(s) are reported. Two five-coordinate [Fe(OEP)(CO)] derivatives have Fe-C = 1.7077(13) and 1.7140(10) A, much shorter than those of six-coordinate [Fe(OEP)(Im)(CO)], although nu(C-O) is 1944-1948 cm(-1). The six-coordinate species [Fe(OEP)(CO)2] has also been studied. The competition for pi-back-bonding of two CO ligands leads to Fe-C distance of 1.8558(10) A and nu(C-O) being increased to 2021 cm(-1). The Mössbauer spectrum has a quadrupole splitting constant of 0 mm/s at 4.2 K, indicating high electronic symmetry.

Low frequency resonance Raman spectra of isolated alpha and beta subunits of hemoglobin and their deuterated analogues.

In an attempt to gain further insight into the nature of the low frequency vibrational modes of hemoglobin and its isolated subunits, a comprehensive study of several different isotopically labeled analogues has been undertaken and is reported herein. Specifically, the resonance Raman spectra, between 200 and 500 cm(-1), are reported for the deoxy and ligated (CO and O2) forms of the isolated alpha and beta subunits containing the natural abundance or various deuterated analogues of protoheme. The deuterated protoheme analogues studied include the 1,3,5,8-C2H3-protoheme (d12- protoheme), the 1,3-C2H3-protoheme (1,3-d6-protoheme), the 5,8-C2H3-protoheme (5,8-d6-protoheme), and the meso-C2H4-protoheme (d4-protoheme). The entire set of acquired spectra has been analyzed using a deconvolution procedure to help correlate the shifted modes with their counterparts in the spectra of the native forms. Interestingly, modes previously associated with so-called vinyl bending modes or propionate deformation modes are shown to be quite sensitive to deuteration of the peripheral methyl groups of the macrocycle, shifting by up to 12-15 cm(-1), revealing their complex nature. Of special interest is the fact that shifts observed for the 1,3-d6- and 5,8-d6-protoheme analogues confirm the fact that certain modes are associated with a given portion of the macrocycle; i.e., only certain modes shift upon deuteration of the 1 and 3 methyl groups, while others shift upon deuteration of the 5 and 8 methyl groups. Compared with the spectra previously reported for the corresponding myoglobin derivatives, the data reported here reveal the appearance of several additional features that imply splitting of modes associated with the propionate groups or that are indicative of greater distortion of the heme prosthetic groups.

Kinetic analysis of semisynthetic peroxidase enzymes containing a covalent DNA-heme adduct as the cofactor.

The reconstitution of apo enzymes with DNA oligonucleotide-modified heme (protoporphyrin IX) cofactors has been employed as a tool to produce artificial enzymes that can be specifically immobilized at the solid surfaces. To this end, covalent heme-DNA adducts were synthesized and subsequently used in the reconstitution of apo myoglobin (aMb) and apo horseradish peroxidase (aHRP). The reconstitution produced catalytically active enzymes that contained one or two DNA oligomers coupled to the enzyme in the close proximity to the active site. Kinetic studies of these DNA-enzyme conjugates, carried out with two substrates, ABTS and Amplex Red, showed a remarkable increase in peroxidase activity of the DNA-Mb enzymes while a decrease in enzymatic activity was observed for the DNA-HRP enzymes. All DNA-enzyme conjugates were capable of specific binding to a solid support containing complementary DNA oligomers as capture probes. Kinetic analysis of the enzymes immobilized by the DNA-directed immobilization method revealed that the enzymes remained active after hybridization to the capture oligomers. The programmable binding properties enabled by DNA hybridization make such semisynthetic enzyme conjugates useful for a broad range of applications, particularly in biocatalysis, electrochemical sensing, and as building blocks for biomaterials.

Synthesis of protoheme IX derivatives with a covalently linked proximal base and their human serum albumin hybrids as artificial hemoprotein.

The simple one-pot reaction of protoporphyrin IX and omega-(N-imidazolyl)alkylamine or O-methyl-L-histidyl-glycine with benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate at room temperature produced a series of protoporphyrin IX species with a covalently linked proximal base at the propionate side-chain. The central iron was inserted by the general FeCl2 method, converting the free-base porphyrins to the corresponding protoheme IX derivatives. Mesoporphyrin IX and diacetyldeuteroporphyrin IX analogues were also prepared by the same procedure. The Fe(II) complexes formed dioxygen (O2) adducts in dimethylformamide at 25 degrees C. Some of them were incorporated into the hydrophobic domain of recombinant human serum albumin (rHSA), providing albumin-heme hybrids (rHSA-heme), which can bind and release O2 in aqueous media (pH 7.3, 25 degrees C). The oxidation process of converting the dioxygenated heme in rHSA to the inactive Fe(III) state obeyed first-order kinetics, indicating that the mu-oxo dimer formation was prevented by the immobilization of heme in the albumin scaffold. The rHSA-heme, in which the histidylglycil tail coordinates to the Fe(II) center, showed the most stable O2 adduct complexes.

Oxidation of carboxylic acids by horseradish peroxidase results in prosthetic heme modification and inactivation.

Hemoproteins are powerful oxidative catalysts. However, despite the diversity of functions known to be susceptible to oxidation by these catalysts, it is not known whether they can oxidize carboxylic acids to carboxylic radicals. We report here that incubation of horseradish peroxidase (HRP) at acidic pH with H(2)O(2) in acetate buffer results in rapid modification of the heme group and loss of catalytic activity. Mass spectrometry and NMR indicate that an acetoxy group is covalently bound to the delta-meso-carbon in the modified heme. A heme with a hydroxyl group on the 8-methyl is also formed as a minor product. These reactions do not occur if protein-free heme and H(2)O(2) are co-incubated in acetate buffer, if the HRP reaction is carried out at pH 7, in the absence of H(2)O(2), or if citrate rather than acetate buffer is used. A similar heme modification is observed in incubations with n-caproic and phenylacetic acids. A mechanism involving oxidation of the carboxyl group to a carboxylic radical followed by addition to the delta-meso-position is proposed. This demonstration of the oxidation of a carboxylic acid solidifies the proposal that a carboxylic radical mediates the normal covalent attachment of the heme to the protein in the mammalian peroxidases and CYP4 family of P450 enzymes. The hemoprotein-mediated oxidation of carboxylic acids, ubiquitous natural constituents, may play other roles in biology.

Effects of systematic peripheral group deuteration on the low-frequency resonance Raman spectra of myoglobin derivatives.

Resonance Raman spectra are reported for a series of systematically deuterated analogues of myoglobin in its deoxy state as well as for its CO and O(2) adducts. Specifically, the myoglobin samples studied are those that have been reconstituted with deuterated protoheme analogues. These include the methine deuterated, protoheme-d4; analogue bearing C(2)H(3) groups at the 1, 3, 5, and 8 positions, protoheme-d12; the species bearing C(2)H(3) groups at the 1 and 3 positions only, 1,3-protoheme-d6; and the species bearing C(2)H(3) groups at the 5 and 8 positions only, 5,8-protoheme-d6. While the results are generally consistent with previously reported data for synthetic metalloporphyrin models and previous studies of labeled heme proteins, the high-quality low-frequency RR data reported here reveal several important aspects of these low-frequency modes. Of special interest is the fact that, using the two d6-protoheme analogues, it is shown that certain modes are apparently localized on particular pyrrole rings, while others are localized on different rings; i.e., several of these low-frequency modes are localized on one side of the heme.