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At least 19 recordsLinked to original sources
PubMed1985

Investigations of cyanide as an infrared probe of hemeprotein ligand binding sites.

The measurement of infrared spectra for cyanide liganded to hemeproteins and hemins has been investigated. The hemeproteins included human methemoglobin A, lamprey methemoglobin, metchlorocruorin, horse metmyoglobin, and horseradish peroxidase. The hemins were dicyanide and monopyridine monocyanide species of deuteroporphyrin IX iron(III) and its 2,4-divinyl(proto) and 2,4-diacetyl derivatives. C-N stretch bands of low intensity detected near 2100 cm-1 exhibit changes in frequency, width, intensity, and isotope shift with changes in cyanide compound structure. Infrared band parameters are particularly sensitive to a change in oxidation state (Fe2+ versus Fe3+) and are affected to a lesser extent by changes in porphyrin ring substituent, ligand trans to the cyanide, and protein structure. Evidence of multiple conformers (i.e. multiple C-N stretch bands) was found for several hemeproteins. The cyanide infrared spectra provide direct evidence for cyanide binding as a metal cyanide (Fe--C identical to N) and against HCN being the ligand in nitrile-like bonding (Fe--N identical to C--H) in all the hemeprotein and hemin cyanides studied. With the reduced horseradish peroxidase cyanide, differences between infrared spectra for D2O and H2O solutions can result from hydrogen bonding between a protein amino acid residue and the distal atom of the cyanide (Fe--C identical to N...H+--R). The binding of cyanide to reduced iron (Fe2+) of a hemeprotein was only observed in the case of the reduced peroxidase. These findings demonstrate that cyanide infrared spectra can not only determine when cyanide is bound to a metalloprotein but can also provide information on how the cyanide is bonded to metal and on characteristics of the ligand binding site.

Animals↗
PubMed1993

Hydrogen peroxide-dependent formation and bleaching of the higher oxidation states of bovine erythrocyte green hemeprotein.

The ferric and ferrous forms of bovine erythrocyte green hemeprotein react with hydroperoxides to form higher oxidation state intermediates with absorbance maxima in the Soret region at 426 and 422 nm, respectively. In the absence of an appropriate reductant, these intermediates undergo rapid bleaching reactions. 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) rapidly reduces the intermediate formed by reaction of ferric green hemeprotein with hydrogen peroxide, thereby preventing bleaching and allowing the rate of the intermediate formation to be calculated from the coupled dye oxidation. This rate constant of 70 M-1 s-1 at 23 degrees C is similar to those determined by bleaching and by direct photometric detection of the intermediate. Dihydroriboflavin rapidly reduces the intermediate formed by reaction of ferrous green hemeprotein with hydrogen peroxide, thereby preventing bleaching and allowing the rate of the intermediate formation to be calculated from the coupled dihydroriboflavin oxidation; the rate constant of 2 x 10(4) M-1 s-1 at 23 degrees C is similar to the value calculated by direct detection of the intermediate. The results demonstrate that, in contrast to the reductase activity of its heme-free form, the green heme form of the protein reacts with hydroperoxides to generate highly unstable peroxide complexes.

Animals↗
PubMed1986

Characterization of the spleen green hemeprotein with magnetic and natural circular dichroism spectroscopy: positive evidence for a myeloperoxidase-type active site.

The green hemeprotein purified from bovine spleen has been characterized with magnetic and natural circular dichroism (MCD and CD) spectroscopy for the first time. The enzyme derivatives studied include the native high-spin ferric form and its high-spin chloride and low-spin cyanide and nitrite complexes, the ligand-free high-spin ferrous form and its low-spin CO adduct, and Compounds II (ferryl iron species) and III (dioxygen adduct). All these enzyme states exhibit MCD spectra that are considerably different from the spectra of analogous complexes of normal heme iron. In particular, the following distinctions have been observed. The sign of the derivative-shaped MCD bands of the high-spin ferric and Compound II forms in the Soret (380-500 nm) region and of the ferrous low-spin and Compound III forms in both the Soret and visible (500-700 nm) regions are opposite to and, except for the high-spin ferric form, are less symmetric than those seen for normal heme iron systems. MCD intensities in the Soret region for the high-spin ferrous and low-spin ferric derivatives are noticeably smaller than those of normal heme proteins by a factor of up to ten. Prominent MCD bands are seen around 450 and 630 nm for the green hemeprotein derivatives; these features are considerably red-shifted (30-50 nm) relative to the analogous transitions observed for normal heme proteins. In contrast to the aforementioned spectral differences, the MCD and CD spectra of the spleen green hemeprotein derivatives are essentially identical to those previously reported for several derivatives of another spectroscopically anomalous heme-type enzyme, myeloperoxidase. This provides strong evidence that the two enzymes have identical prosthetic groups and endogenous axial ligands coordinated to the central iron. The novel MCD features of the green proteins, taken together with previously reported spectroscopic results, are most consistent with the presence of a chlorin-type prosthetic group in both proteins. In addition, the CD spectral similarities suggest that the two green proteins have nearly identical active-site environments.

Animals↗
PubMed1992

Formation of nitrogen oxides and citrulline upon oxidation of N omega-hydroxy-L-arginine by hemeproteins.

HRP catalyzes the oxidation of N omega-hydroxy-L-arginine (NOHA) by H2O2 with formation of citrulline and NO2- with initial rates of about 0.7 and 0.2 nmol per nmol HRP per min. In the same manner, cytochromes P450 from rat liver microsomes catalyze the oxidation of NOHA to citrulline and NO2- by cumylhydroperoxide. Inhibitors of these hemeproteins (N3- and CN- for HRP and miconazole for P450) strongly inhibit both citrulline and NO2- formation. Rates of NOHA oxidation by these hemeproteins markedly decrease with time presumably because of their denaturation by nitrogen oxides and of the formation of hemeprotein-iron-NO complexes. These results suggest that NO (and other nitrogen oxides) could be formed from oxidation of NOHA by other enzymes than NO-synthases.

Animals↗
PubMed1991

Coupling of dihydroriboflavin oxidation to the formation of the higher valence states of hemeproteins.

The reactions between hydrogen peroxide and hemeproteins have been coupled to the oxidation of dihydroriboflavin so as to provide a simple method for measuring the rate constant of hemeprotein peroxidation. Dihydroriboflavin rapidly reduces the higher oxidation states of iron and the hydroxy radicals which are the products of the hemeprotein/hydrogen peroxide reaction. The rapid reduction of these highly reactive compounds prevents the hemeproteins from undergoing irreversible chemical modifications and thus allows the kinetics of peroxidation to be studied. The rate constants at pH 7.2 and 23 degrees C for the peroxidation of horseradish peroxidase, myoglobin, and ferrocytochrome c are found to be 6.2 x 10(6), 7.5 x 10(4), and 8 x 10(3)M-1s-1, respectively. These studies suggest that reduced riboflavin might efficiently protect cells from oxidative damage such as that occurring in inflammation and reperfusion injury.

Animals↗
PubMed1985

Spectroscopic, ligand binding, and enzymatic properties of the spleen green hemeprotein. A comparison with myeloperoxidase.

The bovine spleen green hemeprotein, a peroxidase which exhibits spectrophotometric properties similar to those of granulocyte myeloperoxidase, was purified using an improved method. The ligand affinity of the ferric enzyme was spectroscopically determined using chloride and cyanide as exogenous ligands. The pH dependence of the apparent dissociation constant of the enzyme-chloride complex showed the presence of a proton dissociable group with a pKa value of 4 on the enzyme; chloride binds to the enzyme when this group is protonated with a dissociation constant of 60 microM. The cyanide affinity of the enzyme is also regulated by the group with a pKa value of 4, but in this case cyanide binds to the unprotonated enzyme with a dissociation constant of 0.6 microM; only the protonated, uncharged form of cyanide reacts with the enzyme. Cyanide binding was competitively inhibited by chloride, and chloride binding was also competitively inhibited by cyanide. The EPR spectrum of the resting enzyme exhibited a rhombic high spin signal at g = 6.65, 5.28, and 1.97 with a low spin signal at g = 2.55, 2.32, and 1.82. Upon formation of the chloride complex, the spectrum was replaced with a new high spin EPR signal with g-values of 6.81, 5.04, and 1.95. The cyanide complex showed a low spin EPR signal with g-values of 2.83, 2.25, and 1.66. Examination of the enzymatic activity of the spleen green hemeprotein by following the chlorination of monochlorodimedon has indicated that the enzyme has the same chlorinating activity as myeloperoxidase; the spleen green peroxidase can catalyze the formation of hypochlorous acid from hydrogen peroxide and chloride ion. Comparison of the present data with those of myeloperoxidase has led to the conclusion that the structure of the iron center and its vicinity in spleen green hemeprotein is very similar, if not identical, to that of myeloperoxidase. The spleen enzyme can thus be used as a model to study the active center, and its environment, in myeloperoxidase.

Animals↗
PubMed2006

Hydrogen-bonding conformations of tyrosine B10 tailor the hemeprotein reactivity of ferryl species.

Ferryl compounds [Fe(IV)=O] in living organisms play an essential role in the radical catalytic cycle and degradation processes of hemeproteins. We studied the reactions between H2O2 and hemoglobin II (HbII) (GlnE7, TyrB10, PheCD1, PheE11), recombinant hemoglobin I (HbI) (GlnE7, PheB10, PheCD1, PheE11), and the HbI PheB10Tyr mutant of L. pectinata. We found that the tyrosine residue in the B10 position tailors, in two very distinct ways, the reactivity of the ferryl species, compounds I and II. First, increasing the reaction pH from 4.86 to 7.50, and then to 11.2, caused the the second-order rate constant for HbII to decrease from 141.60 to 77.78 M-1 s-1, and to 2.96 M-1 s-1, respectively. This pH dependence is associated with the disruption of the heme-tyrosine (603 nm) protein moiety, which controls the access of the H2O2 to the hemeprotein active center, thus regulating the formation of the ferryl species. Second, the presence of compound I was evident in the UV-vis spectra (648-nm band) in the reactions of HbI and recombinant HbI with H2O2, This band, however, is completely absent in the analogous reaction with HbII and the HbI PheB10Tyr mutant. Therefore, the existence of a hydrogen-bonding network between the heme pocket amino acids (i.e., TyrB10) and the ferryl compound I created a path much faster than 3.0x10(-2) s-1 for the decay of compound I to compound II. Furthermore, the decay of the heme ferryl compound I to compound II was independent of the proximal HisF8 trans-ligand strength. Thus, the pH dependence of the heme-tyrosine moiety complex determined the overall reaction rate of the oxidative reaction limiting the interaction with H2O2 at neutral pH. The hydrogen-bonding strength between the TyrB10 and the heme ferryl species suggests the presence of a cycle where the ferryl consumption by the ferric heme increases significantly the pseudoperoxidase activity of these hemeproteins.

Ferric Compounds↗
PubMed1968

Spectral studies of iron coordination in hemeprotein complexes: difference spectroscopy below 250 millimicrons.

In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mmu, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mmu, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mmu is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented.

Azides↗
PubMed1995

Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins.

In this short review we show how suitable analysis of the temperature dependence of the optical absorption spectra of metalloproteins can give insight into their stereodynamic properties in the region of the chromophore. To this end, the theory of coupling between an intense allowed electronic transition of a chromophore and Franck-Condon active vibrations of the nearby atoms is applied to the Soret band of hemeproteins to obtain an analytical expression suitable for fitting the spectral profile at various temperatures. The reported approach enables one to separate the various contributions to the overall bandwidth together with the parameters that characterize the vibrational coupling. The thermal behavior of these quantities gives information on the dynamic properties of the active site and on their dependence upon protein structure and ligation state. The Soret band of hemeproteins appears to be coupled to high frequency vibrational modes of the heme group (as already shown by resonance Raman spectroscopy) and to a "bath" of low frequency modes most likely deriving from the bulk of the protein. For the deoxy derivatives inhomogeneous broadening arising from conformational heterogeneity appears to contribute substantially to the linewidth. The data indicate the onset, at temperatures near 180 K, of large scale anharmonic motions that can be attributed to jumping among different conformational substates of the protein.

Animals↗
PubMed1998

Endocytosis by the corneal endothelium. I. Regulation of binding and transport of hemeproteins and peroxidase-conjugated lectins across the tissue.

Binding, internalization, and movement of hemeproteins and peroxidase-conjugated lectins across organ cultured rat corneal endothelia has been investigated. Horseradish peroxidase (HRP) type II, bound to the surface, was minimally internalized and was easily washed off. In contrast, HRP-VI bound and was rapidly internalized. Reaction product was observed in vesicles, endosomes, multivesicular bodies, and extended along the length of the intercellular space (ICS) to Descemet's membrane. Studies at 4 degrees C indicated HRP-VI bound uniformly along the surface in a punctate fashion. Exposure to polylysine or mannose significantly decreased uptake. Other tracers such as HRP-VIII, -IX, catalase, and microperoxidase exhibited limited uptake by the tissue. However, endothelia vigorously internalized soybean agglutinin (SBA)-HRP, and reaction product was found intracellularly and within the ICS at the cell/Descemet's membrane interface. Internalization and the appearance of SBA-HRP within the ICS was diminished following polylysine or mannose treatment. Experiments at 4 degrees C indicated that SBA-HRP binding and uptake were temperature sensitive. Wheat germ agglutinin (WGA)-HRP was also strongly endocytosed and reaction product was visualized within vesicles, endosomes, and multivesicular bodies. Although WGA-HRP reaction product was observed within the ICS, none was detected at the level of Descemet's membrane. The WGA competitive sugar N-acetyl-D-glucosamine, reduced endocytosis, whereas exposure to unlabeled WGA and mannose together reduced uptake. These results indicate endothelia exhibit differential uptake of various hemeproteins and lectins which is dependent on charge, mannose receptors, and appropriate surface sugars.

Animals↗
PubMed1975

An interpretation of the three line EPR spectrum of nitric oxide hemeproteins and related model systems: the effect of the heme environment.

The EPR spectra of the nitric oxide (NO) derivatives of structurally perturbed Fe (II) hemeproteins show various patterns, all of which are characterized by the conspicuous three-line hyperfine splitting due to 14NO, in contrast to that of the native proteins. For the purpose of obtaining structural information from these three line spectra, the model systems were studied, which consist of NO, heme (or TPP-Fe(II), where TTP means alpha, beta, gamma, delta-tetraphenylporphine) and the nitrogenous base, pyridine or quinoline, which, respectively, give the native type or the three line (perturbed type) EPR spectrum. The ring proton paramagnetic shift of quinoline in this system shows that it is not coordinated to NO-TPP-Fe(II) as a normal axial ligand, in contrast to pyridine which gives the shift pattern of the ordinary axial ligation. This observation suggests that in the NO-hemeproteins some perturbations of the protein structure cause the rupture or distortion of the bond between the imidazole nitrogen on the fifth coordination site and the heme iron, resulting in the three line spectrum. The EPR study of the model systems, the pentacoordinated complex, NO-heme and NO-TPP-Fe(II), in various media revealed that the pentacoordinated species indeed exhibits, depending upon its environment, a variety of spectra, which closely reproduce the three line patterns observed in the perturbed proteins and some related model systems. Such spectral variation can be attributed to the difference in the degree of quenching the internal motion and/or the structural heterogeneity caused by molecular environment.

Electron Spin Resonance Spectroscopy↗
PubMed1994

How does NO activate hemeproteins?

NO was reported to activate guanylate cyclase and, recently, prostaglandin H synthase. NO interaction with the heme component in different hemeproteins is determined by ligand property, electronic configuration of the heme iron and the specific effects contributed by the protein structure. It is found that although NO interaction with the free heme provides some common rules of interaction, the consequences of NO binding to different hemeproteins should be dealt with individually.

Hemeproteins↗
PubMed1989

Parameters controlling the kinetics of ferric and ferrous hemeproteins reduction by hydrated electrons.

To clarify the processes of hemeproteins reduction, three classes of these proteins (ferric, ferrous and desFe) were reduced by hydrated electrons generated by pulse radiolysis. Spectral and kinetic investigations were made on alpha hemoglobin chain and myoglobin. Human alpha chain has been chosen to avoid all ferric contaminations and horse ferric myoglobin to eliminate all ferrous protein fractions. We have successively studied the influences of: the iron presence, its oxidation state (II and III), the protein charge and the iron-ligand nature (H2O, OH-, N3- and CN-). For alpha human hemoglobin chain without metallic ion or with ferrous iron, the reduction rates are the same: 1.1 +/- 0.2.10(10) M-1.s-1. In the case of horse ferric myoglobin, the reduction rates depend principally on the protein charge (from pH 6.3 to pH 9.5, the reduction rate of Mb(FeIII)N3- decreases from 2.5 +/- 0.5.10(10) M-1.s-1 to 1.2 +/- 0.2.10(10) M-1.s-1) and are also modulated by the equilibrium constant of the hemeprotein-ligand association (1.2 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)N3- and 0.8 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)CN-, at pH 9.8).

Chemical Phenomena↗
PubMed2006

Oxidative modification of quercetin by hemeproteins.

The ability of a number of hemeproteins to oxidize the flavonoid quercetin has been shown. It was found that quercetin undergoes chemical modification in the presence of cytochrome c, myoglobin, and hemoglobin but not cytochrome b(5). In the range of investigated proteins the most effective oxidant appears to be cytochrome c. Chromatographic analysis of the reaction mixture revealed a number of quercetin oxidation products. The main oxidation product was purified and characterized by means of LC-MS and NMR analyses. It has a dimeric structure similar to the product of quercetin oxidation by horseradish peroxidase and is formed during radical-driven reactions. Our results indicate that a number of hemeproteins can react and modify biologically active flavonoids. However, these reactions might also lead to the generation of active species with deleterious consequences for the cellular macromolecules.

Animals↗
PubMed1999

Cyclooxygenase dependent release of heme from microsomal hemeproteins correlates with induction of heme oxygenase 1 transcription in human fibroblasts.

Induction of heme oxygenase 1 transcription and enzymatic activity is a common response after exposure of cells to various forms of oxidative stress including ultraviolet A radiation (UVA) and hydrogen peroxide. We now show that UVA irradiation or hydrogen peroxide treatment of human skin fibroblasts leads to an immediate release of the heme oxygenase substrate, heme, from microsomal hemeproteins. The release of heme by UVA apparently involves cyclooxygenase activity because it is inhibited by the cyclooxygenase inhibitor indomethacin. We also demonstrate a high degree of correlation between the amount of heme released and the degree of subsequent induction of heme oxygenase 1 transcription following UVA and hydrogen peroxide treatment. We propose that release of heme from microsomal hemeproteins determines the degree of induction of heme oxygenase 1 transcription in human fibroblasts after oxidative stress.

Butylated Hydroxytoluene↗
PubMed1993

Proton NMR of Escherichia coli sulfite reductase: the unligated hemeprotein subunit.

The isolated hemeprotein subunit of sulfite reductase (SiR-HP) from Escherichia coli consists of a high spin ferric isobacteriochlorin (siroheme) coupled to a diamagnetic [4Fe-4S]2+ cluster. When supplied with an artificial electron donor, such as methyl viologen cation radical, SiR-HP can catalyze the six electron reductions of sulfite to sulfide and nitrite to ammonia. Thus, the hemeprotein subunit appears to represent the minimal protein structure required for multielectron reductase activity. Proton magnetic resonance spectra are reported for the first time on unligated SiR-HP at 300 MHz in all three redox states. The NMR spectrum of high spin ferric siroheme at pH 6.0 was obtained for the purpose of comparing its spectrum with that of oxidized SiR-HP. On the basis of line widths, T1 measurements, and 1D NOE experiments, preliminary assignments have been made for the oxidized enzyme in solution. The pH profile of oxidized SiR-HP is unusual in that a single resonance shows a 9 ppm shift over a range of only 3 pH units with an apparent pK = 6.7 +/- 0.2. Resonances arising from the beta-CH2 protons of cluster cysteines have been assigned using deuterium substitution for all redox states. One beta-CH2 resonance has been tentatively assigned to the bridging cysteine on the basis of chemical shift, T1, line width, and the presence of NOEs to protons from the siroheme ring. The observed pattern of hyperfine shifts can be used as a probe to measure the degree of coupling between siroheme and cluster in solution. The cluster iron sites of the resting (oxidized) enzyme are found to possess both positive and negative spin density which is in good agreement with Mossbauer results on frozen enzyme. The NMR spectrum of the 1-electron reduced form of SiR-HP is consistent with an intermediate spin (S = 1) siroheme. Intermediate spin Fe(II) hemes have only been previously observed in 4-coordinate model compounds. However, the amount of electron density transferred to the cluster, as measured by the isotropic shift of beta-CH2 resonances, is comparable to that present in the fully oxidized enzyme despite diminution of the total amount of unpaired spin density available. Addition of a second electron to SiR-HP, besides generating a reduced S = 1/2 cluster with both upfield and downfield shifted cysteine resonances, converts siroheme to the high spin (S = 2) ferrous state.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1981

Permeability of rat choriocapillaris to hemeproteins. Restriction of tracers by a fenestrated endothelium.

The choriocapillaris is the fenestrated capillary bed in the choroid of the eye and is the major blood supply to the retinal pigment epithelium (RPE) and photoreceptor cells. Bruch's membrane (BM) is a multilaminated basement membrane that separates the choriocapillaris from the RPE. In a previous study (Pino RM, Essner E; Cell Tissue Res 208:21, 1980) we found that the choriocapillary endothelium restricted the egress of ferritin from the choriocapillaris. In the present study, hemeproteins were used to further establish the permeability characteristics of this capillary bed. Horseradish peroxidase (Einstein-Strokes radius (ESR), 30 A) rapidly crossed the capillary endothelium (less than 5 min) after intravenous administration and after 5 minutes filled BM and the basal infoldings of the RPE. In contrast, hemoglobin (Hg) (ESR, 32 A) and lactoperoxidase (LP) (ESR, approximately 40 A) are markedly restricted at the level of endothelial diaphragmed fenestrae, channels, and intercellular junctions. Little vesicular transport of these proteins was observed. The reaction product of the two hemeprotein activities was not demonstrable in BM for up to 30 min after injection; relatively low levels were detected after 75 min. HG and LP appear to be further restricted by BM, since their reaction products were not demonstrable between the RPE basal infoldings at this time. Catalase (ESR, 52 A) activity was not detected in BM for up to 4 hr after injection. These results indicate that the rat choriocapillary endothelium, unlike the fenestrated endothelia lining other vascular beds, substantially restricts the passage of large tracer molecules.

Animals↗
PubMed1989

Biomimetic chemistry of hemeproteins.

Hemeproteins play important physiological roles for an oxygen metabolism in living organisms. Their functions are divided to three main groups, i) hemoglobins, myoglobins and the cytochromes which function by transporting and storing dioxygen and electrons; ii) catalase and peroxidases which are activated by hydrogen peroxide, iii) cytochrome oxidase and cytochrome P450 both of which bind dioxygen and use the dioxygen as an electron sink or by partially reducting the dioxygen to make a powerful oxidizing agent. The hemeproteins included to group ii) have been established to produce a reaction intermediate, oxo-ferry (Fe(IV] pi-cation radical, during the catalytic cycle. Recent model studies to mimic cytochrome P450 catalyzed-reaction have shown that the intermediate retains an activated oxygen and functions as a powerful oxidizing agent in the monoxygenase reaction. In this article, the properties of the intermediate and the role in cytochrome P450 monoxygenase reaction is summarized.

Chemical Phenomena↗