The interaction of methemoglobin and modified poly-(hydroxyethylmethacrylates) studied by spectroscopic methods

The interaction between methemoglobin (MetHb) and macroporous matrices on the basis of polymethacrylates was investigated by means of optical and e.p.r. spectroscopy. The spectroscopic data show that the adsorption of MetHb to imidazole-containing matrices occurs by complex formation between matrix-bound imidazole and the iron of the prosthetic group, with all 4 polypeptide chains of the MetHb molecule being included in the interaction. The adsorption to hydrophobic side chains containing matrices leads, via the protein-matrix interaction, to considerable disturbances of iron protoporphyrin IX in equilibrium or formed from protein-contacts, which are of general importance with respect to the functional variablity and control, respectively, of iron porphyrins in hemoproteins. In case of matrix containing n-hexyl groups deoxyHb is oxidized by O2 to MetHb, instead of being oxygenated to HbO2. Not all prosthetic groups are able to bind N-3. With the increase in hydrophobicity of the matrix a conformational change is enforced leading in the beta-chains to the direct interaction between iron and sulfur of cysteine (beta-cys 92), as it is proved in all cytochrome P-450 and other model compounds.     

Proton NMR study of myoglobin reconstituted with 3, 7-diethyl-2, 8-dimethyl iron porphyrin: remarkable influence of peripheral substitution on heme rotation

The iron complex of 3,7-diethyl-2,8-dimethylporphyrin was incorporated into horse heart apomyoglobin to investigate the influence of peripheral substitution on artificial heme rotation. The hyperfine-shifted 1H NMR spectrum of the reconstituted deoxymyoglobin (rMb) revealed the proximal imidazole N-H resonance at 82.5 ppm to indicate the formation of the Fe--N (His93) bond. The pyrrole-protons of the hemin of myoglobin in the absence of external ligand appeared as four resonances between -10 and -18 ppm, indicating a mainly low-spin ferric hemin, with a ligated distal histidine (His64). This also indicates the lost of the symmetry of the hemin, according to an absence of free rotation of the prosthetic group. The 1H NMR spectrum of reconstituted rMbCO revealed a set of four pyrrole-protons and a set of four meso-protons. Accordingly, the prosthetic group without acid side chains interacts specifically with the surrounding globin showing a unique heme orientation in the 1H NMR time-scale, despite the presence of only four alkyl substituents on the porphine ring. This also suggests that two ethyl groups are large enough to avoid the free rotation movement of the heme.     

Possible Role of Metal(II) Octacyanomolybdate(IV) in Chemical Evolution: Interaction with Ribose Nucleotides

We have proposed that double metal cyanide compounds (DMCs) might have played vital roles as catalysts in chemical evolution and the origin of life. We have synthesized a series of metal octacyanomolybdates (MOCMos) and studied their interactions with ribose nucleotides. MOCMos have been shown to be effective adsorbents for 5′-ribonucleotides. The maximum adsorption level was found to be about 50 % at neutral pH under the conditions studied. The zinc(II) octacyanomolybdate(IV) showed larger adsorption compared to other MOCMos. The surface area seems to important parameter for the adsorption of nucleotides. The adsorption followed a Langmuir adsorption isotherms with an overall adsorption trends of the order of 5′-GMP > 5′-AMP > 5′-CMP > 5′-UMP. Purine nucleotides were adsorbed more strongly than pyrimidine nucleotides on all MOCMos possibly because of the additional binding afforded by the imidazole ring in purines. Infrared spectral studies of adsorption adducts indicate that adsorption takes place through interaction between adsorbate molecules and outer divalent ions of MOCMos.     

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH₂CH(X)COOCH₃ > RCH₂CH(X)CONH₂ > RCH₂CH(X)COOH > RCH₂CH₂X > RCH₂CH(X)CH₂OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS^?) is the reactive species, but only minor differences emerged between different free thiolates.     

Active site topology of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase (CYP51) and its G310D mutant (cytochrome P-450SG1).

Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.     

Synthesis and biological evaluation of new non-imidazole H3-receptor antagonists of the 2-aminobenzimidazole series

A novel series of non-imidazole H(3)-receptor antagonists was developed, by chemical modification of a potent lead H(3)-antagonist composed by an imidazole ring connected through an alkyl spacer to a 2-aminobenzimidazole moiety (e.g., 2-[[3-[4(5)-imidazolyl]propyl]amino]benzimidazole), previously reported by our research group. We investigated whether the removal of the imidazole ring could allow retaining high affinity for the H(3)-receptor, thanks to the interactions undertaken by the 2-aminobenzimidazole moiety at the binding site. The imidazole ring of the lead was replaced by a basic piperidine or by a lipophilic p-chlorophenoxy substituent, modulating the spacer length from three to eight methylene groups; moreover, the substituents were moved to the 5(6) position of the benzimidazole nucleus. Within both the 2-alkylaminobenzimidazole series and the 5(6)-alkoxy-2-aminobenzimidazole one, the greatest H(3)-receptor affinity was obtained for the piperidine-substituted compounds, while the presence of the p-chlorophenoxy group resulted in a drop in affinity. The optimal chain length was different in the two series. Even if the new compounds did not reach the high receptor affinity shown by the imidazole-containing lead compound, it was possible to get good H(3)-antagonist potencies with 2-aminobenzimidazoles having a tertiary amino group at appropriate distance.     

Transferrin-bipyridine iron transfer mediated by haemoproteins

Rabbit reticulocyte cytosol was able to mediate transferrin-bipyridine iron transfer in the presence of ATP. The cytoplasmic factor responsible for the mediation of iron transfer was identified as haemoglobin. Other cytoplasmic proteins and the membrane fraction were ineffective. Human α and β subunits and human myoglobin were over three times more effective than human haemoglobin A. Carbon monoxide strongly inhibited the mediation of iron transfer. Oxidation of haemoglobin abolished it but methaemoglobin could be reactivated with NADH, even when azide was bound to the haem iron.Neither globin nor haem alone were able to mediate iron transfer, even when NADH was present. Together, the reconstituted methaemoglobin A could be reactivated with NADH.Although the physiological significance of this phenomenon is not clear, the involvement of haemoproteins in intracellular iron metabolism seems likely.     

Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent

Soret excitation resonance Raman spectroscopy has been used to characterize dimeric human leukocyte myeloperoxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) and monomeric bovine spleen green haemoprotein. The spectra of the two proteins, under the same conditions of iron valence and ligation, are essentially identical. Owing to strong symmetry reduction effects, the spectra are more complex than usually observed for haemoproteins. It is possible, however, to assign the high-frequency vibrations and, from these assignments, to determine structural features of the iron chromophores. In the resting protein, the iron adopts a six-coordinate high-spin configuration in both proteins; cyanide addition produces six-coordinate low-spin species, and in the ferrous enzymes the iron appears to be five-coordinate and high-spin. The proteins are stable to laser excitation and do not photoreduce under illumination. No evidence is found for unusual peripheral substituents, such as formyl or protonated Schiff's base group, in conjugation with the main chromophore in the native protein. The vibrational data are consistent with an iron chlorin chromophore, although other electronic effects, in addition to those produced by porphyrin ring reduction, are necessary to account for the optical properties of the proteins. The similarity in Raman spectra for myeloperoxidase and green haemoprotein indicates that the two iron sites in myeloperoxidase are equivalent.     

Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.     

Molecular dynamics simulations of phenylimidazole inhibitor complexes of cytochrome P450 cam

Molecular dynamics simulations have been performed on three phenylimidazole inhibitor complexes ofP450 _cam, utilizing the X-ray structures and the AMBER suite of programs. Compared to their corresponding optimized X-ray structures, very similar features were observed for the 1-phenylimidazole (1-PI) and 2-phenylimidazole (2-PI) complexes during a 100 ps MD simulation. The 1-PI inhibitor binds as a Type II complex with the imidazole nitrogen as a ligand of the heme iron. Analysis of the inhibitor-enzyme interctions during the MD simulations reveals that electrostatic interactions of the imidazole with the heme and van der Waals interactions of the phenyl ring with nearby hydrophobic residues are dominant. By contrast, 2-PI binds as a Type I inhibitor in the substrate binding pocket, but not as a ligand of the iron. The interactions of this inhibitor are qualitatively different from that of the Type II 1-PI, being mainly electrostatic/H-bonding interactions with a bound water and polar residues. Although the third compound, 4-PI, in common with 1-PI, also binds as a Type II inhibitor, with one nitrogen of the imidazole as a ligand to the iron, the MD average binding orientation deviates significantly from the X-ray structure. The most important changes observed include: (1) the rotation of the imidazole ring of this inhibitor by about 90° to enhance electrostatic interactions of the imidazole NH group with the carbonyl group of LEU244, and (2) the rotation of the carbonyl group of ASP251 to form a H-bond with VAL254. An analysis of the H-bonding network surrounding this substrate in the optimized crystal structure revealed that there is no H-bonding partner either for the free polar NH group in the imidazole ring of 4-phenylimidazole or for the polar carbonyl group of the nearby ASP251 residue. The deviation of the dynamically averaged inhibitor-enzyme structure of the 4-PI complex from the optimized crystal structure can therefore be rationalized as a consequence of the optimization of the electrostatic interactions among the polar groups.     

Haem as a multifunctional regulator

Haem has long been known as the prosthetic group of haemoproteins such as haemoglobin, catalase and the cytochromes. Its biosynthesis is regulated by feedback mechanisms that ensure its adequate production but prevent its overaccumulation, which is highly deleterious as diseases such as porphyrias attest. However, recent years have seen rapid strides in our understanding of how haem (or more accurately haemin, its oxidized form) itself acts as an intracellular regulator of a variety of other metabolic pathways for systems that utilize oxygen.     

QSAR by LFER model of cytotoxicity data of anti-HIV 5-phenyl-1-phenylamino-1H-imidazole derivatives using principal component factor analysis and genetic function approximation

Cytotoxicity data of anti-HIV 5-phenyl-1-phenylamino-1H-imidazole derivatives were subjected to quantitative structure-activity relationship (QSAR) study using linear free energy related (LFER) model of Hansch using electronic (Hammett sigma), hydrophobicity (pi) and steric (molar refractivity and STERIMOL L, B1, B2, B3 and B4) parameters of phenyl ring substituents of the compounds, along with appropriate indicator variables. Principal component factor analysis (FA) was used as the data-preprocessing step to identify the important predictor variables contributing to the response variable and to avoid collinearities among them. The generated multiple linear regression (MLR) equations were statistically validated using leave-one-out technique. Genetic function approximation (GFA) was also used on the same data set to develop QSAR equations, which produced the same best equation as obtained with FA-MLR. The final equation is of acceptable statistical quality (explained variance 80.2%) and predictive potential (leave-one-out predicted variance 74%). The analysis explores the structural and physicochemical contributions of the compounds for cytotoxicity. A thiol substituent at 2 position of the imidazole nucleus decreases cytotoxicity when compared to the corresponding unsubstituted congener. Presence of hydrogen bond donor group at meta position of the phenyl ring present at 5 position of the imidazole nucleus also reduces cytotoxicity. Additionally, absence of any substituent at 2 and 3 positions of the phenyl ring of 1-phenylamino fragment reduces the cytotoxicity. The negative coefficient of sigmap indicates that presence of electron-withdrawing substituents at the para position of the phenyl ring of the 1-phenylamino fragment is not favourable for the cytotoxicity. Again, lipophilicity of meta substituents of the 5-phenyl ring increases cytotoxicity. The coefficients of molar refractivity (MRm) and STERIMOL parameters for meta substituents (Lm, B1m and B4m) of the phenyl ring of 1-phenylamino fragment indicate that the length, width and overall size of meta substituents are conducive factors for the cytotoxicity.     

Exploring the molecular determinants of substrate-selective inhibition of cyclooxygenase-2 by lumiracoxib

Lumiracoxib is a substrate-selective inhibitor of endocannabinoid oxygenation by cyclooxygenase-2 (COX-2). We assayed a series of lumiracoxib derivatives to identify the structural determinants of substrate-selective inhibition. The hydrogen-bonding potential of the substituents at the ortho positions of the aniline ring dictated the potency and substrate selectivity of the inhibitors. The presence of a 5′-methyl group on the phenylacetic acid ring increased the potency of molecules with a single ortho substituent. Des-fluorolumiracoxib (2) was the most potent and selective inhibitor of endocannabinoid oxygenation. The positioning of critical substituents in the binding site was identified from a 2.35 Å crystal structure of lumiracoxib bound to COX-2.     

Design,synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH₂-imidazole or CH₂-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC?=?12.5?μg/mL, 17a 50?μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2?Å. A model generated from a 1.5?Å crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.     

Assignment of the axial ligands of the haem in milk lactoperoxidase using magnetic circular dichroism spectroscopy

The absorption and MCD spectra of ferric lactoperoxidase from milk and its cyanide and fluoride derivatives have been measured in the near infrared and visible wavelength regions both at room temperature and at 4.2 K. By comparison with the MCD spectra of haemoproteins of known axial ligation, which also contain protohaem IX, it has been possible to arrive at suggestions for the axial ligation in lactoperoxidase. At room temperature oxidized lactoperoxidase has the haem iron in the high-spin state, and the results indicate that the proximal ligand of the haem iron is a histidine imidazole and that the sixth ligand is probably a carboxylate ion. At 4.2 K oxidized lactoperoxidase converts almost totally to a low-spin form, changing the sixth ligand to a histidine imidazole, which is in the imidazolate form.     

State of heme in heme c systems: Cytochrome c and heme c models

Polarized resonance Raman spectra of horse heart ferricytochrome c as a function of pH in the range 1.0–12, in the presence of the extrinsic ligands imidazole, cyanide, and azide, and in 4 M urea, are reported, as are resonance Raman spectra of heme undecapeptide in the presence of imidazole, pH 6.8 and pH 2.0, and with cyanide at pH 6.8. The range of investigation is 140–1700 cm^?1, using the 5145-, 4880-, and 4579-Å excitations. The spectra have been analyzed in terms of complexity, sensitivity, and the conformation-heme energetics of the systems. The state of heme in various forms is analyzed with regard to heme energetics, core size, nature of planarity, and coordination configuration. All low-spin forms of heme c systems, cytochrome c, and heme models are concluded to be hexacoordinated, in-plane heme iron systems. The effect of the location of the heme in the protein environment is found to be a slight expansion of the porphyrin core, ～0.01 Å, while the covalent linkage of heme to protein and a mixed nature of axial coordination configuration seem to have little effect on the energetics of the heme group. Complex formation with extrinsic ligand, imidazole, cyanide, or azide, results in a slight contraction of the heme core. The formation of cytochrome c form IV, the alkaline form, is shown to follow a process with apK _a of about 8.4, and similarly, acidic form II is created following the prior formation of an intermediate form with apK _a of about 3.6. The precursor to form IV is interpreted as containing perturbation of the pyrrol rings, whereas the precursor to the acidic form seems to reflect alteration of the energetics of the C_αC_{m α} structures of the heme group. The acidic form of heme undecapeptide is a hexacoordinated high-spin heme with an estimated displacement of 0.25 Å from the heme plane. The pH 2 form of cytochrome c is also a hexacoordinated high-spin form with two weak axial ligands, but iron is in the plane of the porphyrin ring.     

Control of haem synthesis by feedback inhibition on human-erythrocyte δ-aminolaevulate dehydratase

The action of haem, haemoglobin and other haemoproteins on crude and purified δ-aminolaevulate dehydratase of human erythrocyte is described. The results show a feedback inhibition by haem of porphyrin synthesis at the level of erythrocyte δ-aminolaevulate dehydratase. Some kinetic characteristics of this inhibition are described.     

Non-equilibrium state of a monomeric insect haemoglobin induced by γ-irradiation and detected by Mössbauer spectroscopy

The met-aquo form of the monomeric insect haemoglobin CTT III has been investigated by Mössbauer spectroscopy before and after reduction with thermolyzed electrons at low temperature. The native met haemoglobin dissolved in water and water/glycerol mixtures, respectively, exhibits in the range of pH 5.8 to 9.0 high-spin iron(III). The electronic state of the haemoglobin is not affected by the solvent conditions. In water/glycerol γ-irradiation at 77 K results in the reduction of the haem iron by thermolyzed electrons. Due to this process, the hexacoordinated high-spin iron(III) is transformed into a hexacoordinated low-spin iron(II). This latter complex is a transition state which changes into the high-spin iron(II) state of the deoxyhaemoglobin when increasing the temperature. Thus, a kinetically stabilized non-equilibrium state of the haemoglobin exists at low temperature which relaxes with increasing temperature and finally reaches the equilibrium state to form deoxyhaemoglobin. This transition occurs at T > 190 K and corresponds with drastic changes in the temperature dependence of the Lamb-Mössbauer factor. Both effects indicate an alteration of the intramolecular flexibility of the haemoglobin.     

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

 The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. Received: 17 July 1996 / Accepted: 28 November 1996     

Localization of repetitive and unique DNA sequences neighbouring the rabbit beta-globin gene

The structure of oxymyoglobin has been refined at 1·6 Å resolution, using diffractometer data collected at ?12 °C. The crystallographic R factor is 0·159, and the atomic positions are known to 0·1 Å accuracy in internal segments of the molecule.The iron atom lies 0·22(3) Å from the plane of the porphyrin, 0·25 Å closer than in deoxymyoglobin, and the F helix has moved by a similar amount. Oxygen binds to the iron in a bent, end-on arrangement, with FeOO = 115(5) ° and FeO = 1·83(6) Å. The mean FeN(porphyrin) bond length is 1·95(6) Å, 0·08 Å shorter than in deoxymyoglobin, but the difference is not significant compared to the experimental error. FeN_ε(His8F) is 2·07(6) Å, the same as in model compounds. Movements of the haem, iron, F helix and FG corner on oxygenation are similar to those found in the T-R state transition in haemoglobin, but are smaller in magnitude.