Exploring the Strength of the H‐Bond in Synthetic Models for Heme Proteins: The Importance of the N−H Acidity of the Distal Base

The distal hydrogen bond (H‐bond) in dioxygen‐binding proteins is crucial for the discrimination of O₂ with respect to CO or NO. We report the preparation and characterization of a series of Zn^II porphyrins, with one of three meso‐phenyl rings bearing both an alkyl‐tethered proximal imidazole ligand and a heterocyclic distal H‐bond donor connected by a rigid acetylene spacer. Previously, we had validated the corresponding Co^II complexes as synthetic model systems for dioxygen‐binding heme proteins and demonstrated the structural requirements for proper distal H‐bonding to Co^II‐bound dioxygen. Here, we systematically vary the H‐bond donor ability of the distal heterocycles, as predicted based on pK_a values. The H‐bond in the dioxygen adducts of the Co^II porphyrins was directly measured by Q‐band Davies‐ENDOR spectroscopy. It was shown that the strength of the hyperfine coupling between the dioxygen radical and the distal H‐atom increases with enhanced acidity of the H‐bond donor.     

A role for highly conserved carboxylate, aspartate-140, in oxygen activation and heme degradation by heme oxygenase-1.

Heme oxygenase (HO) catalyzes the oxygen-dependent degradation of heme to biliverdinIXalpha, CO, and free iron ion via three sequential monooxygenase reactions. Although the distinct active-site structure of HO from cytochrome P450 families suggests unique distal protein machinery to activate molecular oxygen, the mechanism and the key amino acid for the oxygen activation have not been clear. To investigate the functionality of highly conserved polar amino acids in the distal helix of HO-1, we have prepared alanine mutants: T135A, R136A, D140A, and S142A, and found drastic changes in the heme degradation reactions of D140A. In this paper, we report the first evidence that D140 is involved in the oxygen activation mechanism in HO-1. The heme complexes of HO mutants examined in this study fold and bind heme normally. The pK(a) values of the iron-bound water and autoxidation rates of the oxy-form are increased with R136A, D140A, and S142A mutations, but are not changed with T135A mutation. As the wild-type, T135A, R136A, and S142A degrade heme to verdohemeIXalpha with H(2)O(2) and to biliverdinIXalpha with the NADPH reductase system. On the other hand, D140A heme complex forms compound II with H(2)O(2), and no heme degradation occurs. For the NADPH reductase system, the oxy-form of D140A heme complex is accumulated in the reaction, and only 50% of heme is degraded. The stopped flow experiments suggest that D140A cannot activate iron-bound dioxygen and hydroperoxide properly. To investigate the carboxylate functionality of D140, we further replaced D140 with glutamic acid (D140E), phenylalanine (D140F), and asparagine (D140N). D140E degrades heme normally, but D140N shows reactivity similar to that of D140A. D140F loses heme degradation activity completely. All of these results indicate that the carboxylate at position 140 is essential to activate the iron-bound dioxygen and hydroperoxide. On the basis of the present findings, we propose an oxygen activation mechanism involving the hydrogen-bonding network through the bridging water and D140 side chain.     

Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state. The ferrous heme protein reacts with carbon monoxide to form a (1-methyl-histidine)-Fe(II)(heme)-CO complex; however, reaction with dioxygen leads to autoxidation and ferric heme dissociation. These results indicate that negative protein design can be used to generate a five-coordinate heme within a maquette scaffold.     

Blue-LED Synthesis of Pummerer's Ketones by Peroxidase-Lignin-Catalyzed Oxidative Coupling of Substituted Phenols

Pummerer's ketones resembling the tricyclic scaffold of bioactive natural substances were synthesized by blue-LED driven Horseradish Peroxidase oxidative coupling of substituted phenols in 2-methyltetrahydrofuran by using meso-tetraphenylporphyrin as photosensitizer and dioxygen as primary oxidant. The application of functionalized lignin nanoparticles as a renewable and efficient platform for the immobilization of the enzyme extended the effectiveness of the overall process to heterogeneous catalysis under buffer limiting conditions.     

Oxidation of 5,15-Dioxaporphyrin: Its Generality and Novelty as an Oxaporphyrin Analogue

5,15-Dioxaporphyrin ( DOP ) is a novel meso-oxaporphyrin analogue and exhibits unique 20π-antiaromaticity, unlike its mother congener of 18π-aromatic 5-oxaporphyrin, commonly known as its cationic iron complex called verdohem, which is a key intermediate of heme catabolism. To reveal its reactivities and properties as an oxaporphyrin analogue, the oxidation of tetra-β-arylated DOP ( DOP-Ar₄ ) was explored in this study. Stepwise oxidation from the 20π-electron neutral state was achieved, and the corresponding 19π-electron radical cation and 18π-electron dication were characterized. Further oxidation of the 18π-aromatic dication resulted in the formation of a ring-opened dipyrrindione product by hydrolysis. Considering a similar reaction of verdoheme to ring-opened biliverdin in the heme degradation in nature, the current result consolidates the ring-opening reactivity of oxaporphyrinium cation species.     

Hydrophobic behavior,ROS production,and heme degradation of hemoglobin upon interaction with <Emphasis Type="Italic">n</Emphasis>-alkyl sulfates

The oxygen-containing free radical species form upon interaction of amphiphilic substances such as sodium dodecyl sulfate and hemoglobin. Under these conditions, hemoglobin is converted to methemoglobin and simultaneously results in heme degradation. Since heme is located in a hydrophobic moiety of hemoglobin, we hypothesized that other hydrophobic substances or amphiphilic xenobiotics can dispose hemoglobin to oxidative stress. Here this hypothesis was tested by investigating heme degradation of hemoglobin during interaction with n-alkyl sulfates. This was accomplished using UV–Vis and fluorescence spectroscopy, chemometric analysis, and chemiluminescence methods. We determined whether a relationship exists between the alkyl tail length (surfactant hydrophobicity) of n-alkyl sulfate homologues, reactive oxygen species (ROS) production, and heme degradation pattern of hemoglobin. We also proposed a mechanism for these types of interactions and induction of heme degradation. Our results indicated that hemoglobin structural–functional changes including globin denaturation are the key factors in starting the heme degradation process, and heme degradation product patterns were dependent on each alkyl sulfate. However, the number of fluorescent components (heme degradation products) was independent of the alkyl sulfate type. The reason for this phenomenon was the mechanism of reaction in which the amount of hydrogen peroxide was changed with each homologue, but the mechanism of degradation remained the same. Thus, an increase in hydrophobic chain length of the surfactants correlated with the enhanced ROS production and heme degradation of hemoglobin.     

Spectroscopic detection and quantification of heme and heme degradation products

Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532, and 752?nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin, and bilirubin. Concentrations in the low micromolar range (down to 3?μmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (SERS) detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA, and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers where the analytes have to compete for the colloid. With the help of statistical analysis, it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights into their interaction.

Spectroscopic characterization and electrocatalytical activity of tetrapyridylporphyrins intercalated into hydrated vanadium(V) oxide

The intercalation ofmeso-tetrapyridylporphyrins into hydrated vanadium(V) pentoxide has been studied using X-ray diffraction; FTIR, electronic and Mössbauer spectroscopy; cyclic voltammetry and spectroelectrochemistry. The intercalation compound containing iron tetrapyridylporphyrin exhibits pronounced electrocatalytical activity in the reduction of molecular dioxygen, reflecting an enhanced reactivity of the catalyst in a confined medium.     

Self-assembly of meso-mono-4-pyridyltriphenylporphyrinatoiron(ii) in sublimed layers. Interaction with molecular oxygen

Low-temperature (T = 80 K) interaction of the sublimed layers of meso-mono-4-pyridyltriphenylporphyrinatoiron(ii) (FeMPyTPP) with dioxygen was studied by IR and electronic absorption spectroscopies. Unlike the meso-tetraphenylporphyrinatoiron(ii) (FeTPP) layers, coordination of O₂ with FeMPyTPP produces extra-complexes of two types: in the first complex one of the axial sites is free, while in the second complex an axial site is occupied by the pyridine group of the adjacent molecule. The results obtained indicate self-assembly of the FeMPyTPP molecules through coordination bonds between the iron atom and pyridine group of the adjacent molecule in the layer. The bonding of O₂ by the sublimed FeMPyTPP layers differs substantially from that by the FeTPP layers, which rapidly loose their ability of oxygen bonding at room temperature.     

Electrocatalytic Reactions by an Iron Porphyrin/Polypyrrole Modified Electrode Monitored by Electrochemical Quartz Crystal Microbalance

Electropolymerization of pyrrole in aqueous solution is monitored by electrochemical quartz crystal microbalance (EQCM) during potential scan. The film formation process on the electrode is reflected by the frequency decrease for the quartz oscillator as a function of time. The film growth rate is greatly enhanced in the presence of iron(III) meso-tetrakis(3-sulfonatomesityl)porphyrin, which carries 3- of charge. The metalloporphyrin is trapped and remains intact in the polypyrrole films. The ion transport property through the iron porphyrin/polypyrrole film is the reverse of that for pure polypyrrole. Electroreduction of dioxygen and electrooxidation of alkenes are parallel to those in the solution state.     

Electronic structures of heme a of cytochrome c oxidase in the redox states—Charge density migration to the propionate groups of heme a

The electronic structures of heme a of cytochrome c oxidase in the redox states were studied, using hybrid density functional theory with a polarizable continuum model and a point charge model. We found that the most stable electronic configurations of the d electrons of the Fe ion are determined by the orbital interactions of the d orbitals of the Fe ion with the π orbitals of the porphyrin ring and the His residues. The redox reaction of the Fe ion influences the charge density on the formyl group through the π conjugation of the porphyrin ring. In addition, we found the charge transfer from the Fe ion to the propionate group of heme a in the redox change despite the lack of the π‐conjugation. We elucidated that the charge propagation originates from the heme a structure itself and that the origin of the charge delocalization to the heme propionate is the orbital interactions between the d orbital of the Fe ion and the p orbitals of the carboxylate part of the heme propionate via the π conjugation of the porphyrin ring and the σ* orbital of the C? C bond of the propionate group. The electrostatic effect by surrounding proteins enhances the charge transfer from the Fe ion to the propionate group. These results indicate that heme propionate groups serve electron mediators in electron transfer as well as electrostatic anchors, and that proteins surrounding the active site reinforce the congenital abilities of the cofactors. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Synthesis of meso-Alkyl-Substituted Norcorrole–NiII Complexes and Conversion to 5-Oxaporphyrins(2.0.1.0)

The synthesis of antiaromatic Ni^II–norcorroles having primary, secondary, and tertiary alkyl groups at the reactive meso-positions was attempted. Reductive coupling of a Ni^II–dipyrrin precursor provided Ni^II–meso-dihexylnorcorrole, which underwent substantial degradation on silica gel. Introduction of tert-butyl groups was unsuccessful due to the difficult preparation of the corresponding dipyrrin precursor. Meanwhile, Ni^II–norcorroles with isopropyl and cyclohexyl groups were isolated as stable molecules under ambient conditions. Furthermore, we found that oxidation of Ni^II–meso-dialkylnorcorroles with hydrogen peroxide in the presence of sodium carbonate gave Ni^II–5-oxaporphyrins(2.0.1.0). In contrast, oxidation of Ni^II–meso-dimesitylnorcorrole under the same reaction conditions gave 10-oxaporphyrin(1.1.1.0). The contrasting reactivity can be attributed to the steric congestion around the meso-positions.     

Efficient photodissociation of O2 from synthetic heme and heme/M (M = Fe, Cu) complexes

Single wavelength excitation (lambdaex = 355 or 532 nm) of low-temperature stabilized (198 K) synthetic heme-dioxygen and heme-dioxygen/M complexes, where M = copper or iron in a non-heme environment, results in the dissociation of dioxygen as indicated by the generation of the ferrous heme (Soret band, 427 nm) and the bleaching of the ferric-superoxide (FeIII(O2-)) 410-nm Soret band in the transient absorption difference spectrum. Dioxygen rebinds to the four heme complexes studied with comparable rate constants ( approximately 6-9 x 105 M-1 s-1). However, the quantum yield for complete dissociation of O2 from our simplest heme-O2 complex (F8)FeIII(O2-) (phi = 0.60) is higher than the other complexes measured (phi = approximately 0.2-0.3) as well as that for oxy-myoglobin (phi = 0.3).     

Antibacterial photoactivity and photosensitized oxidation of phenols with meso-tetra-(4-benzoate, 9-phenanthryl)-porphyrin and its metal complexes (Zn and Cu)

We performed the synthesis and characterization of meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin and its Zn and Cu complexes. Synthesis of meso-tetra-(4-benzoate-9-phenanthryl)-porphyrin was carried out by dry gaseous HCl, meso-tetra-(4-carboxyphenyl)-porphyrin, and 9-phenantrol in tetrahydrofuran. The preparation of metal complexes was carried out using the method of the acetates. All porphyrins were characterized by FT-IR, NMR (¹H and ¹³C), MS, UV–visible, and fluorescence spectroscopy. Their photophysical properties: photostability, fluorescence quantum yields, energy transfer, and generation of singlet oxygen were determined and compared with the meso-tetraphenylporphyrin. Photochemical studies on their effectiveness as photosensitizers were performed through photo-oxidation of alcohols, phenol, and 2-naphthol. Higher efficiency of degradation was obtained with photostable TB9FPCu. The antibacterial photoactivity assay was tested against Escherichia coli and its viability was measured by chemiluminescence. The highest inactivation levels were obtained by ester TB9FP and Zn complex. The properties of the photosensitizer and its efficiency vary as a result of modifying its structure. The results obtained show that the efficiency of a photosensitizer depends on multiple factors. Thus, we can say that the copper complex is efficient in degradation of alcohol, while the metal-free porphyrin is better for antibacterial applications.     

Dendrimers with Porphyrin Cores: Synthetic models for globular heme proteins

Dendritic iron porphyrins were synthesized as functional mimics of globular electron-transfer heme proteins. The cascade molecules 1 · Zn ? 3 Zn of first to third generation were obtained starting from the (meso-diarylporphyrin) zinc 6 · Zn which contains four carboxylate arms for attachment of the poly(ether-amide) dendritic branches by peptide-coupling methodology (Scheme 1). Generation 3 compound 3 · Zn with 108 methyl-carboxylate end groups has a molecular weight of 19054. D, and computer modeling suggests that its structure is globular and densely-packed, measuring ca. 4 nm in diameter and, therefore, similar in dimensions to the electron-transfer protein cytochrome-c. Starting from the generation 1 poly(carboxylic acid) 11 · Zn and the generation 2 analog 12 · Zn the dendritic Zn^II porphyrins 4 · Zn and 5 · Zn , respectively, were obtained by esterification with triethyleneglycol monomethyl ether (Schemes 3 and 4). Demetallation followed by insertion of Fe^II and in situ oxidation afforded the water-soluble dendritic iron porphyrins 4 FeCl and 5 FeCl . The electrochemical behavior of esters 1 · Zn ? 3 · Zn in organic solvents changed smoothly with increasing dendritic generation (Table 1). Progressing from 1 · Zn to 3 · Zn in THF, the first porphyrin-centered oxidation and reduction potentials become more negative by 320 and 210mV, respectively. These changes were attributed to strong microenvironmental effects imposed on the electroactive core by the densely packed dendritic surroundings. The electrochemical properties of 4 · FeCl and 5 · FeCl were investigated by cyclic voltammetry in both CH₂Cl₂ and H₂O (Tables 2 and 3). Progressing from 4 · FeCl to 5 · FeCl in CH₂Cl₂, the redox potential of the biologically relevant Fe^III/Fe^II couple remained virtually unchanged, whereas in aqueous solution, 5 FeCl exhibited a potential 420 mV more positive than did 4 FeCl. The large difference between these potentials in H₂O was attributed to differences in solvation of the core electrophore. Whereas the relatively open dendritic branches in 4 · Fecl do not impede access of bulk solvent to the central core, the densely packed dendritic superstructure of 5 · FeCl significantly reduces contact between the heme and external solvent. As a result, the more charged Fe^III state is destabilized relative to Fe^II, and the redox potential is strongly shifted to a more positive value.     

Role of heme types in heme-copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.     

Highly selective isolation and purification of heme proteins in biological samples using multifunctional magnetic nanospheres

Magnetic particles with suitable surface modification are capable of binding proteins selectively, and magnetic separations have advantages of rapidity, convenience, and high selectivity. In this paper, new magnetic nanoparticles modified with imidazolium ionic liquid (Fe₃O₄@SiO₂@ILs) were successfully fabricated. N‐Methylimidazolium was immobilized onto silica‐coated magnetic nanoparticles via γ‐chloropropyl modification as a magnetic nanoadsorbent for heme protein separation. The particle size was about 90 nm without significant aggregation during the preparation process. Hemoglobin as one of heme proteins used in this experiment was compared with other nonheme proteins. It has been found that the magnetic nanoparticles can be used for more rapid, efficient, and specific adsorption of hemoglobin with a binding capacity as high as 5.78 mg/mg. In comparison with other adsorption materials of proteins in the previous reports, Fe₃O₄@SiO₂@ILs magnetic nanoparticles exhibit the excellent performance in isolation of heme proteins with higher binding capacity and selectivity. In addition, a short separation time makes the functionalized nanoparticles suitable for purifying unstable proteins, as well as having other potential applications in a variety of biomedical fields.     

Reversible binding of nitric oxide and carbon-carbon bond formation in a meso-hydroxylated heme

Exposure of a pyridine solution of (py)2Fe(OEPO) (1) (OEPO is the trianion of octaethyloxophlorin, py is pyridine) at 22 degrees C to nitric oxide under the strict exclusion of dioxygen results in a color change from green to red-brown and the formation of {(py)(ON)Fe(OEPO)}2 (2). Monitoring the reaction by 1H NMR spectroscopy shows that the reaction is reversible. The air-sensitive product {(py)(ON)Fe(OEPO)}2 has been isolated and characterized by X-ray crystallography. NO binding is accompanied by association of two hemes through the formation of a new C-C bond between two meso carbon atoms on the porphyrin periphery. The macrocyclic ligand has a ruffled distortion that positions the C5-O1 group with its short C=O bond distance (1.237(3) A) in close proximity to the NO ligand. Since further attack upon 1 by O2 during heme degradation involves reaction in the vicinity of the oxygenated meso position, the positioning of the NO ligand so that the O2...C5 distance is only 3.100(3) A presents a highly suggestive model for the next stage of attack upon the heme periphery.     

Development of the Peripheral Functionalization Chemistry of meso-Free Corroles

In contrast to the extensive development of the meso-functionalization of porphyrins, that of corroles had rarely been explored until the development of practical synthetic methods for meso-free corroles in 2015. The ready availability of meso-free corroles opened up meso-functionalization chemistry of corroles, giving rise to successful synthesis of various meso-substituted corroles such as meso-halogen, meso-nitro, meso-amino, meso-oxo, and meso-iminocorroles as well as meso–meso-linked corrole dimers and corrole tapes. In some cases, 2NH corroles exist as stable or transient radical species. The impact of meso-functionalization on the structures, electronic properties, optical characteristics, and aromaticity of corroles are highlighted in this Minireview.     

A trans-ortho asymmetrically di-strapped metalloporphyrin integrating three key structural features of ligand in heme

Heme responsible for the dioxygen fixation, transport and conversion is a metalloporphyrin complex highly dependent on its diverse geometry of ligand. In this work, a trans-ortho-di-strapped zinc porphyrin with dome-like deformation was synthesized by thermodynamically controlling the formation of trans-precursor of porphyrinogen. Its single-crystal structure demonstrated that the asymmetric treatment of porphyrin achieves three goals of creating two secondary coordination sphere (SCS) bulks, maintaining a unique dome deformation, and making atomic out-of-plane deviation. In this way, this metallic complex integrates at least three key features of the pocket structure, the differentiated axial ligations, and the ring distortion, making it an ideal heme analog.