Modification of the heme·heme oxygenase system with iron and cobalt tetrasulfonated phthalocyanines

Modification of heme·heme oxygenase by iron(III) and cobalt(II) tetrasulfonated phthalocyanines has been performed. New compounds have been isolated and their properties have been investigated by difference spectroscopy, electrophoresis, molecular weight estimation, electron paramagnetic resonance (EPR) and carboxymethylation at histidyl groups. Spectrophotometric titration data indicate the ratio of the reagents in this process to be 1:1. The visible absorption spectra show the main peak at 650 nm for the iron compound and 682 nm for the cobalt one. Electrophoresis and molecular weight estimation show both complexes to be monomers. Cobalt(II) tetrasulfonated phthalocyanine, under aerobic conditions with heme oxygenase protein, undergoes autooxidation to the cobalt(III) complex, as has been proved by EPR and spectroscopic data. Iron and cobalt phthalocyanine modified heme·heme oxygenase with excess dithionite is reduced at the phthalocyanine ligand. In the presence of oxygen, the reduction product transforms into oxygenated Fe(III)Lheme oxygenase or Co(III)heme oxygenase, respectively. Reduction of the iron(III) model complex with ascorbic acid under anaerobic conditions leads to degradation of the phthalocyanine moiety, while Co(III)heme oxygenase with ascorbic acid is reduced to Co(II)Lheme oxygenase. As has been shown by carboxymethylation of the heme oxygenase protein at the histidine residues, the predominant binding site of both phthalocyanine complexes is the heme-binding histidyl residue. There is evidence that there is a second binding site with lower affinity towards Co(II)L on the heme oxygenase protein. Iron and cobalt tetrasulfonated phthalocyanines are not able to displace heme from the heme·heme oxygenase complex. In this reaction the iron complex undergoes degradation and the cobalt one gives a hybrid compound with heme·heme oxygenaseHeme oxygenase protein complexes with iron and cobalt tetrasulfonated phthalocyanines do not exhibit activity in their oxidative degradation.     

Interaction of porphyrins with heme proteins – a brief review

In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.     

Structural characterization of a carbon monoxide adduct of a heme–DNA complex

Abstract  

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3′-terminal G-quartet of the DNA though a π–π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π–π stacking interaction between the pseudo-C ₂-symmetric heme and the C ₄-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C ₂ axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe–CO bond in the complex is similar to that of the Fe–CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.     

Kinetics of the reconstitution of hemoglobin from semihemoglobins α and β with heme

Kinetics of the reconstitution of hemoglobin from semihemoglobins and with hemin dicyanide have been investigated using three kinds of stopped-flow technique (Soret absorption, fluorescence quenching of tryptophan, and Soret CD). The semihemoglobins and are occupied by heme in the and chains, respectively, the other chain being heme-free. Based on the kinetic results, the following scheme for the reconstitution is proposed; First, hemin dicyanide enters the pocket-like site of the apo chains. Second, in semihemoglobin , the CN-ligand in the fifth coordination position of iron is replaced by the imidazole ring of the proximal His immediately after the heme insertion. In contrast, semihemoglobin changes its conformation after the heme insertion, and this is followed by the ligand replacement. Finally, the partial structure changes induced by the ligand replacement propagate onto the whole molecule and the final conformation is attained. The results indicate that semihemoglobin retains a more rigid and organized structure, and more closely approaches its final structure than does semihemoglobin . Correspondence to: Y. Kawamura-Konishi     

Low-cost equilibrium unfolding of heme proteins using 2 μl samples

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure–function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2 μl of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b₅, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed.     

NirF is a periplasmic protein that binds d₁ heme as part of its essential role in d₁ heme biogenesis

The cytochrome cd? nitrite reductase from Paracoccus pantotrophus catalyses the one electron reduction of nitrite to nitric oxide using two heme cofactors. The site of nitrite reduction is the d? heme, which is synthesized under anaerobic conditions by using nirECFD-LGHJN gene products. In vivo studies with an unmarked deletion strain, ΔnirF, showed that this gene is essential for cd? assembly and consequently for denitrification, which was restored when the ΔnirF strain was complemented with wild-type, plasmid-borne, nirF. Removal of a signal sequence and deletion of a conserved N-terminal Gly-rich motif from the NirF coded on a plasmid resulted in loss of in vivo NirF activity. We demonstrate here that the product of the nirF gene is a periplasmic protein and, hence, must be involved in a late stage of the cofactor biosynthesis. In vitro studies with purified NirF established that it could bind d? heme. It is concluded that His41 of NirF, which aligns with His200 of the d? heme domain of cd?, is essential both for this binding and for the production of d? heme; replacement of His41 by Ala, Cys, Lys and Met all gave nonfunctional proteins. Potential functions of NirF are discussed.     

Does the reduction of c heme trigger the conformational change of crystalline nitrite reductase?

The structures of nitrite reductase from Paracoccus denitrificans GB17 (NiR-Pd) and Pseudomonas aeruginosa (NiR-Pa) have been described for the oxidized and reduced state (Fül?p, V., Moir, J. W. B., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M. C., Mathieu, M., Cutruzzolà, F., Bourgeois, D., Fül?p, V., Hajdu, J., Brunori, M., Tegoni, M., and Cambillau, C. (1997) Structure 5, 1157-1171; Nurizzo, D., Cutruzzolà, F., Arese, M., Bourgeois, D., Brunori, M., Cambillau, C. , and Tegoni, M. (1998) Biochemistry 37, 13987-13996). Major conformational rearrangements are observed in the extreme states although they are more substantial in NiR-Pd. The four structures differ significantly in the c heme domains. Upon reduction, a His17/Met106 heme-ligand switch is observed in NiR-Pd together with concerted movements of the Tyr in the distal site of the d1 heme (Tyr10 in NiR-Pa, Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62 in NiR-Pa, 99-116 in NiR-Pd). Whether the reduction of the c heme, which undergoes the major rearrangements, is the trigger of these movements is the question addressed by our study. This conformational reorganization is not observed in the partially reduced species, in which the c heme is partially or largely (15-90%) reduced but the d1 heme is still oxidized. These results suggest that the d1 heme reduction is likely to be responsible of the movements. We speculate about the mechanistic explanation as to why the opening of the d1 heme distal pocket only occurs upon electron transfer to the d1 heme itself, to allow binding of the physiological substrate NO2- exclusively to the reduced metal center.     

Translational inhibition by heme of the synthesis of hepatic δ-aminolevulinate synthase in a cell-free system

Synthesis of δ-aminolevulinate synthase in a rabbit reticulocyte lysate system directed by total polysomes from the liver of allylisopropylacetamide-treated rats was studied with the combined use of [³H]leucine and a specific rabbit antibody. The protein synthesis observed in the cell-free system employed represented mainly the peptide chain elongation and its termination rather than the net synthesis involving initiation. Synthesis of δ-aminolevulinate synthase in this cell-free system was inhibited progressively with the increased addition of hemin; the synthesis was reduced to about 40% by about 30 μM hemin. Synthesis of total protein, however, was not significantly affected by the addition of hemin. The data obtained suggest that heme inhibits a peptide chain elongation step in the synthesis of δ-aminolevulinate synthase.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

On the preparation and mössbauer properties of some heme peptides of cytochrome c

The preparations of a series of peptides derived from horse heart cytochrome c by proteolytic digestions are reported. The Mössbauer spectra of the hexa- and nonapeptides are reported here for the first time and compared with those of the undecapeptide and the parent cytochrome. The nona- and undecapeptides have Mössbauer spectra similar to that of ferricytochrome c and would appear to be useful models for the iron environment. As with free hemes, pyridine may act as a reducing agent, and thus we wish to record a caveat against the use of this compound during peptide preparation.     

Inhibitors of the heme oxygenase - carbon monoxide system: on the doorstep of the clinic?

The past decade has seen substantial developments in our understanding of the physiology, pathology, and pharmacology of heme oxygenases (HO), to the point that investigators in the field are beginning to contemplate therapies based on administration of HO agonists or HO inhibitors. A significant amount of our current knowledge is based on the judicious application of metalloporphyrin inhibitors of HO, despite their limitations of selectivity. Recently, imidazole-based compounds have been identified as potent and more selective HO inhibitors. This 'next generation' of HO inhibitors offers a number of desirable characteristics, including isozyme selectivity, negligible effects on HO protein expression, and physicochemical properties favourable for in vivo distribution. Some of the applications of HO inhibitors that have been suggested are treatment of hyperbilirubinemia, neurodegenerative disorders, certain types of cancer, and bacterial and fungal infections. In this review, we address various approaches to altering HO activity with a focus on the potential applications of second-generation inhibitors of HO.     

Isoniazid and rifampicin inhibit allosterically heme binding to albumin and peroxynitrite isomerization by heme–albumin

Human serum heme–albumin (HSA-heme) displays globin-like properties. Here, the allosteric inhibition of ferric heme [heme-Fe(III)] binding to human serum albumin (HSA) and of ferric HSA–heme [HSA-heme-Fe(III)]-mediated peroxynitrite isomerization by isoniazid and rifampicin is reported. Moreover, the allosteric inhibition of isoniazid and rifampicin binding to HSA by heme-Fe(III) has been investigated. Data were obtained at pH 7.2 and 20.0 °C. The affinity of isoniazid and rifampicin for HSA [K ₀ = (3.9 ± 0.4) × 10⁻⁴ and (1.3 ± 0.1) × 10⁻⁵ M, respectively] decreases by about 1 order of magnitude upon heme-Fe(III) binding to HSA [K _h = (4.3 ± 0.4) × 10⁻³ and (1.2 ± 0.1) × 10⁻⁴ M, respectively]. As expected, the heme-Fe(III) affinity for HSA [H ₀ = (1.9 ± 0.2) × 10⁻⁸ M] decreases by about 1 order of magnitude in the presence of saturating amounts of isoniazid and rifampicin [H _d = (2.1 ± 0.2) × 10⁻⁷ M]. In the absence and presence of CO₂, the values of the second-order rate constant (l _on) for peroxynitrite isomerization by HSA-heme-Fe(III) are 4.1 × 10⁵ and 4.3 × 10⁵ M⁻¹ s⁻¹, respectively. Moreover, isoniazid and rifampicin inhibit dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) in the absence and presence of CO₂. Accordingly, isoniazid and rifampicin impair in a dose-dependent fashion the HSA-heme-Fe(III)-based protection of free l-tyrosine against peroxynitrite-mediated nitration. This behavior has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow’s site I (i.e., the binding pocket of isoniazid and rifampicin) or protrudes into the heme-Fe(III) cleft, depending on ligand binding to Sudlow’s site I or to the FA1 pocket, respectively. These results highlight the role of drugs in modulating heme-Fe(III) binding to HSA and HSA-heme-Fe(III) reactivity.     

NMR assignments of cd-HO, a 24 kDa heme oxygenase from Corynebacterium diphtheria

We are employing a number of selective in vitro and in vivo methods including NMR to screen compounds that bind to heme oxygenases from pathogenic bacteria. We report the nearly complete HN, N, CO, Cα and Cβ chemical shift assignments of a 215-amino acid HO from Corynebacterium diphtheria in three forms, apo cd-HO-G135A, apo cd-HO and CO-bound ferrous holo cd-HO; these assignments will enable us to identify residues on cd-HO that are perturbed upon binding to selected compounds, and to help with the development of inhibitors specific to the bacterial proteins.     

Reduction of oxaporphyrin ring of CO-bound α-verdoheme complexed with heme oxygenase-1 by NADPH-cytochrome P450 reductase

Heme oxygenase (HO) catalyses the degradation of heme to biliverdin, carbon monoxide (CO) and ferrous iron via three successive monooxygenase reactions, using electrons provided by NADPH-cytochrome P450 reductase (CPR) and oxygen molecules. For cleavage of the oxaporphyrin ring of ferrous α-verdoheme, an intermediate in the HO reaction, involvement of a verdoheme π-neutral radical has been proposed. To explore this hypothetical mechanism, we performed electrochemical reduction of ferrous α-verdoheme-rat HO-1 complex under anaerobic conditions. Upon binding of CO, an O₂ surrogate, the midpoint potential for one-electron reduction of the oxaporphyrin ring of ferrous α-verdoheme was increased from −0.465 to −0.392 V vs the normal hydrogen electrode. Because the latter potential is close to that of the semiquinone/reduced redox couple of FAD in CPR, the one-electron reduction of the oxaporphyrin ring of CO-bound verdoheme complexed with HO-1 is considered to be a thermodynamically likely process. Indeed the one-electron reduced species, [Fe^II(verdoheme•)], was observed spectroscopically in the presence of CO in both NADPH/wild-type and FMN-depleted CPR systems under anaerobic conditions. Under physiological conditions, therefore, it is possible that O₂ initially binds to the ferrous iron of α-verdoheme in its complex with HO-1 and an electron is subsequently transferred from CPR, probably via FAD, to the oxaporphyrin ring.     

Thermoregulatory response to hypoxia after inhibition of the central heme oxygenase–carbon monoxide pathway

(1) Centrally acting carbon monoxide (CO) seems to play thermoregulatory actions, but no report exists about its role in hypoxia-induced anapyrexia. (2) CO arises from the catabolism of heme by heme oxygenase (HO), an enzyme that is overexpressed during hypoxia. Thus, we tested the hypothesis that the central HO–CO pathway modulates hypoxia-induced anapyrexia by means of intracerebroventricular injection of the HO inhibitor ZnDPBG. (3) Core temperature (T_C) of awake rats was determined by biotelemetry. ZnDPBG did not alter basal T_c, but it exacerbated hypoxia-induced anapyrexia, indicating that the central HO–CO pathway is a modulator of hypoxia-induced anapyrexia, probably preventing excessive decreases in T_c.