A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.     

A spectrophotometric method for estimating hemin in biological systems

Hemin chlorides exhibit two absorption maxima in the Soret region, one at about 360-380 nm (S' band) and the other between 400 and 430 nm (S band). We present here a simple and fast spectrophotometric assay to determine concentration of hemin between 1.15 and 9.20 microM employing the Soret region (S' band) as a reference. In this method the hemin is quantitatively extracted from biological materials by acidified chloroform. By recording the absorbance of the chloroform extract at its maximum peak at 388, 450, and 330 nm and applying the correction formula A(c)=2A388-(A450+A330), a very good linear correlation between the A(c) and the concentration of hemin is attained. The method can be used to estimate hemin in the presence of protein (0.06-5.00 mg/ml) and porphyrin (0.19-2.97 microM). Compared with the pyridine hemochromogen method, the assay reported here is highly reproducible, with 15- to 30-fold more sensitivity, and it allows the quantification of four times lower hemin concentrations.     

Mechanism of low-density lipoprotein oxidation by hemoglobin-derived iron

Excellular hemoglobin is an extremely active oxidant of low-density lipoproteins (LDL), a phenomenon explained so far by different mechanisms. In this study, we analyzed the mechanism of met-hemoglobin oxidability by comparing its mode of operation with other hemoproteins, met-myoglobin and horseradish peroxidase (HRP) or with free hemin. The kinetics of met-hemoglobin activity toward LDL lipids and protein differed from that of met-myoglobin and HRP, both quantitatively and qualitatively. Those differences were further clarified by analyzing heme transfer from the above-mentioned hemoproteins to LDL. It appeared that met-hemoglobin transferred most of its hemin to LDL, and the presence of H(2)O(2) accelerated the process. In contrast, met-myoglobin partially released hemin, but only in the presence of H(2)O(2), while HRP could not transfer heme at all. The minor amount of hemin transferred from met-myoglobin to LDL sufficed to trigger ApoB oxidation, forming covalent aggregates via inter-bityrosines. This indicated that heme bound to high affinity site(s) is responsible for oxidation. LDL components providing the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra revealed that the high affinity site of monomeric hemin is located on the LDL protein, ApoB. The complex heme-CO-ApoB underwent instantaneous oxidation to hemin-ApoB, and the bound hemin then slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented LDL oxidation by trapping hemoprotein transferable heme. We concluded that met-hemoglobin exerts its oxidative activity on LDL via transfer of heme, which serves as a vehicle for iron insertion into the LDL protein, leading to formation of atherogenic LDL aggregates.     

Hemin/nitrite/H2O2 induces brain homogenate oxidation and nitration: effects of some flavonoids

Oxidative injury has been implicated in the pathogenesis of numerous neurodegenerative diseases. Recently, it has been found that with the existence of hydrogen peroxide and nitrite, hemin catalyzes protein nitration. We hypothesize under certain pathological conditions, hemin catalyzed protein nitration may happen in the brain. In this paper, the effects of three flavonoids, i.e. quercetin, catachin and baicalein on hemin/nitrite/H2O2 induced brain homogenate oxidation and nitration were studied. The results showed that hemin/nitrite/H2O2 system could effectively induce brain homogenate protein oxidation and nitration. Quercetin, catachin and baicalein dose-dependently inhibited hemin/nitrite/H2O2 system-induced protein nitration in a dose-dependent manner, the inhibition of protein nitration was in the order of quercetin>catachin>baicalein. These compounds also inhibited hemin/H2O2 system-induced lipid peroxidation, the inhibition order was baicalein >quercetin>catachin. However, these flavonoids showed marginal effect on hemin/nitrite/H2O2 system caused protein oxidation and thiol oxidation. The inhibition activities of flavonoids on hemin/nitrite/H2O2 system-induced protein nitration may closely relate to their radical scavenging activities, since the inhibition order of protein nitration is the same as the radical scavenging order. These results indicate hemin/nitrite/H2O2 system induces different types of oxidative assault on bio-molecules. Flavonoids could act as antioxidants inhibiting ROS and RNS caused brain damage.     

Purification and biochemical characterization of a heme containing peroxidase from the human parasite P. falciparum

A peroxidase (30 kDa) has been purified from the human malaria parasite Plasmodium falciparum to its homogeneity. The protein is a dimer of 15 kDa subunit as evident from SDS-PAGE and MALDI-TOF mass analysis. The antibodies developed against the purified protein cross-react selectively with this protein present in parasite lysate. It is a heme containing peroxidase [R/Z value (A408/A278)=2.33] showing characteristic heme spectra with Soret peak at 408 nm and visible peaks at 536 and 572 nm. Analysis of Soret spectra in presence or absence of cyanide or azide reveals that iron of heme is in Fe-III state. Circular dichroism spectral analysis establishes that this protein contains mainly alpha-helix (60-70%). H2O2 interacts with the heme moiety of the enzyme as evidenced by optical difference spectroscopy and spectral studies indicate the formation of catalytically active peroxidase-H2O2 complex (Soret peak at 413 nm) to exhibit peroxidase activity. During the erythrocytic stages of its life cycle, the parasite is exposed to oxidative stress. As the parasite is susceptible to oxidative stress, this peroxidase may offer antioxidant role by scavenging endogenous H2O2.     

Crotonobetaine reductase from Escherichia coli consists of two proteins

Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrophoretic homogeneity from a cell-free extract of E. coli O44 K74. The purified protein shows l(-)-carnitine dehydratase activity and its N-terminal amino acid sequence is identical to the caiB gene product from E. coli O44 K74. The relative molecular mass of CI has been determined to be 86100. It is composed of two identical subunits with a molecular mass of 42600. The isoelectric point of CI was found to be 4.3. CII was purified from an overexpression strain in one step by ion exchange chromatography on Fractogel EMD TMAE 650(S). The N-terminal amino acid sequence of CII shows absolute identity with the N-terminal sequence of the caiA gene product, i.e. of the postulated crotonobetaine reductase. The relative molecular mass of the protein is 164400 and it is composed of four identical subunits of molecular mass 41500. The isoelectric point of CII is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecular-mass effector described for the l(-)-carnitine dehydratase is also necessary for crotonobetaine reductase activity. Monoclonal antibodies were raised against the two components of crotonobetaine reductase.     

Mechanism of horseradish peroxidase-catalyzed conversion of iodine to iodide in the presence of EDTA and H2O2

EDTA not only blocks the horseradish peroxidase (HRP)-catalyzed iodide oxidation to I-3 but also causes an enzymatic conversion of oxidized iodine species to iodide (Banerjee, R. K., De, S. K., Bose, A. K., and Datta, A. G. (1986) J. Biol. Chem. 261, 10592-10597). The EDTA effect on both of these reactions can be withdrawn with a higher concentration of iodide and not with H2O2. Spectral studies indicate a possible interaction of EDTA with HRP as evidenced by the formation of modified compound 1 with H2O2 at 416 nm instead of 412 nm in the absence of EDTA. EDTA causes a hypochromic effect on HRP at 402 nm which undergoes the bathochromic red shift to 416 nm by H2O2. The addition of iodide to the 416 nm complex causes the reappearance of the Soret band of HRP at 402 nm. Among various EDTA analogues tested, N-N-N'-N'-tetramethylethylenediamine (TEMED) is 80% as effective as EDTA in the conversion of I-3 to iodide and produces a spectral shift of HRP similar to EDTA. Interaction of EDTA with HRP is further indicated by the hyperchromic effect of HRP and H2O2 on the absorption of EDTA at 212 nm. The addition of oxidized iodine species produces a new peak at 230 nm due to formation of iodide. EDTA at a higher concentration can effectively displace radioiodide specifically bound to HRP indicating its interaction at the iodide-binding site. The enzyme, after radioiodide displacement with EDTA, shows a characteristic absorption maximum at 416 nm on the addition of H2O2, indicating that EDTA is bound with the enzyme. Both positive and negative circular dichroism spectra of HRP and the HRP.H2O2 complex, characteristic of heme absorption, are altered by EDTA, suggesting an EDTA-induced conformational change at or near the heme region. This is associated with a change of affinity of heme toward H2O2 and azide. It is postulated that EDTA interacts at the iodide-binding site of the HRP inducing a new conformation that blocks iodide oxidation but is suitable to convert iodine to iodide by a redox reaction with H2O2.     

Histidine 20, the crucial proximal axial heme ligand of bacterial heme oxygenase Hmu O from Corynebacterium diphtheriae

The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His --> Ala Hmu O mutants. Reconstituted with hemin, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy. EPR of the NO-bound ferrous heme-Hmu O mutant complexes reveals His(20) as the proximal heme ligand in Hmu O, and this is confirmed by resonance Raman results from the ligand-free ferrous heme-H20A. All eight His --> Ala mutants bind hemin stoichiometrically, proving that none of the histidines is essential for hemin-Hmu O formation. However, His(20) is crucial to Hmu O catalysis. Its absence by point mutation has inhibited the conversion of hemin to biliverdin. The ferric heme-H20A complex is pentacoordinate. Resonance Raman of the CO-bound ferrous heme-H20A corroborates this and reveals an Fe-C-O bending mode, delta(Fe-C-O), the first reported for a pentacoordinate CO-bound hemeprotein. The appearance of delta(Fe-C-O) in C. diphtheriae Hmu O H20A but not mammalian HO-1 mutant H25A indicates that the heme environment between the two heme oxygenases is different.     

Interaction of antimalarial drugs with hemin

Hemin (ferriprotoporphyrin IX) is shown to form complexes with the chloroquine class of antimalarial drugs. The Soret band of hemin becomes optically active upon the addition of chiral drugs. Results on the hemin-induced quenching of the fluorescence of chloroquine are consistent with the formation of a 2:1 hemin:drug complex with a formation constant of 1.4 x 10(7) at 298 K. Also a direct comparison of the drug-treated and drug-free parasites themselves, by the noninvasive photoacoustic spectroscopic method, reveals an in vivo interaction between endogenous hemin and the added drug.     

The role of superoxide radicals in lactoperoxidase-catalysed H2O2-metabolism and in irreversible enzyme inactivation

Irreversible inactivation of lactoperoxidase in the presence of excess H2O2 has been investigated. Serial overlay absorption spectra of the Soret region show that the rate and total amount of enzyme inactivation depend on the proton concentration. Perhydroxyl or superoxide radicals (HO.2 or O-2) cannot be established as the inactivating species in this mechanism, but they influence the rate of reconversion of the intermediate lactoperoxidase-compound III back to the resting ferric form of the enzyme.     

Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

Absence of a detectable intermediate in the compound I formation of horseradish peroxidase at ambient temperature

A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H(2)O(2) by HRP is significantly faster than 1 x 10(4) s(-1).     

Generation of excited species catalyzed by horseradish peroxidase or hemin in the presence of reduced glutathione and H2O2

Horseradish peroxidase (HRP) (EC 1.11.1.7) catalyzes the oxidation of reduced glutathione. This reaction is accompanied by light emission, which is attributed to the generation of singlet oxygen. The chemiluminescence is directly related to thiyl radical formation, as deduced from the correlation between the time course of HRP-compound II formation and light emission in the presence of different amounts of H2O2. Superoxide dismutase has an inhibitory effect on the chemiluminescence without affecting the HRP-compound II formation. This indicates the direct involvement of superoxide radicals in the production of photoemissive species. Replacement of HRP by hemin is also accompanied by chemiluminescence.     

Hemin-promoted peroxidation of red cell cytoskeletal proteins

Hemin-induced crosslinking of the erythrocyte membrane proteins was analyzed at three levels: (i) whole membranes, (ii) integrated or dissociated cytoskeletons, and (iii) isolated forms of the three main cytoskeletal proteins, spectrin, actin, and protein 4.1. Addition of H2O2 and hemoglobin to resealed membranes from without did not affect any of the membrane proteins. Hemin that can transport across the membrane induced, in the presence of H2O2, crosslinking of protein 4.1 and spectrin. Both free hemin and hemoglobin added with H2O2 induced crosslinking of integer cytoskeletons and mixtures of isolated cytoskeletal proteins, but hemin was always more active. Of the three major cytoskeletal proteins, spectrin and protein 4.1 were most active while the participation of actin was only minor. The yield of crosslinked products was increased in all reaction mixtures with pH, with an apparent pK above 9.0. Replacement of H2O2 by phenylhydrazine and tert-butyl hydroperoxide resulted in crosslinking of the same proteins, but with lower activity than H2O2. Bityrosines, which were identified by their specific fluorescence emission characteristics, were formed in reaction mixtures containing hemin and hydrogen peroxide and either spectrin or protein 4.1, but not actin. On the basis of fact that bityrosines were revealed only in reaction mixtures that produced protein adducts, formation of intermolecular bityrosines was analyzed to be involved in crosslinking of the cytoskeletal proteins. Since the levels of membrane-intercalated hemin are correlated with aggregation of membrane proteins, it is suggested that the peroxidative properties of hemin are responsible for its toxicity.     

A mechanism for oxygen exchange between ligated oxometalloporphinates and bulk water

Oxygen exchange between high-valent metal-oxo complexes and bulk water has been monitored for nonligated model porphyrins (hemin, FeTDCPPS, MnTMPyP) and the axially ligated microperoxidase-8 (MP-8). Exchange extents up to 90% were measured for MP-8 in spite of the presence of an axial histidine ligand and accompanied by the formation of nonlabelled H(2)O(2) from H(2)(18)O(2). These results point to the existence of a mechanism for oxygen exchange between the high-valent iron-oxo complex and the solvent different from the so-called "oxo-hydroxo tautomerism." Regeneration of the primary oxidant, H(2)O(2), and oxygen exchange by axially ligated porphyrins can be explained by a mechanism involving the reversibility of compound I formation.     

Peroxidative crosslinking of myosins.

The effect of myoglobin, free hemin and H2O2 on myosins from heart and skeletal muscle was studied. SDS-gel electrophoresis revealed that each agent caused intermolecular thiol crosslinking of both myosins dissociable by excess of beta-mercaptoethanol. In the simultaneous presence of H2O2 and myoglobin or H2O2 and free hemin, myosin formed covalent aggregates undissociable by beta-mercaptoethanol and therefore assessed to formation of non S-S inter molecular covalent bonds. The latter aggregates are suggested to result from pairing of myosin radicals formed by the H2O2 induced ferryl iron state in myoglobin, free hemin or hemo-myosin.     

Characterization of MbrC involved in bacitracin resistance in Streptococcus mutans

Deferoxamine (DFO), an FDA-approved iron chelator used for treatment of iron poisoning, affects bacteria as iron availability is intimately connected with growth and several virulence determinants. However, little is known about the effect on oral pathogens. In this study, the effect of DFO on Porphyromonas gingivalis, a major periodontopathogen which has an essential growth requirement for hemin (Fe(3+)-protoporphyrin IX), was evaluated. The viability of P. gingivalis W83 was not affected by 0.06-0.24 mM DFO, whereas the doubling time of the bacterium was considerably prolonged by DFO. The inhibitory effect was evident at earlier stages of growth and reduced by supplemental iron. UV-visible spectra using the pigments from P. gingivalis cells grown on blood agar showed that DFO inhibited μ-oxo bisheme formation by the bacterium. DFO decreased accumulation and energy-driven uptake of hemin by P. gingivalis. Antibacterial effect of H(2)O(2) and metronidazole against P. gingivalis increased in the presence of DFO. Collectively, DFO is effective for hemin deprivation in P. gingivalis suppressing the growth and increasing the susceptibility of the bacterium to other antimicrobial agents such as H(2)O(2) and metronidazole. Further experiments are necessary to show that DFO may be used as a therapeutic agent for periodontal disease.     

Formation and reduction of a ''peroxy'' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.     

Hemin inhibits cyclooxygenase-2 expression through nuclear factor-kappa B activation and ornithine decarboxylase expression in 12-O-tetradecanoylphorbol-13-acetate-treated mouse skin

Inflammation induced by various stimuli has been found to be associated with increased risk for most types of human cancer. Inflammation facilitates the initiation of normal cells, as well as the growth of initiated cells and their progression to malignancy through production of proinflammatory cytokines and diverse reactive oxygen/nitrogen species. These also activate the signaling molecules that are involved in inflammation and carcinogenesis. Our previous studies have demonstrated that hemin inhibited 7,12-dimethylbenz[a]anthracene (DMBA)-induced bacterial mutagenesis and oxidative DNA damage, reduced the level of DNA-DMBA adduct and 12-O-tetradecanoylphorobl-13-acetate (TPA)-induced tumor formation in DMBA-initiated ICR mouse skin, and inhibited myeloperoxidase and ornithine decarboxylase (ODC) activity and H(2)O(2) formation in TPA-treated mouse skin. In the present study, to further elucidate the molecular mechanisms underlying the chemopreventive activity of hemin, its effect on the expression of ODC and cyclooxygenase (COX)-2, and the activation of nuclear factor-kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPKs) regulating these proteins were explored in mouse skin with TPA-induced inflammation. Topically applied hemin inhibited ear edema and epidermal thickness in mice treated with TPA. Pretreatment with hemin reduced the expression of ODC and COX-2, and also reduced NF-kappaB activation in TPA-stimulated mouse skin. In addition, hemin suppressed the TPA-induced activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK in a dose-dependent manner. Taken together, hemin inhibited TPA-induced COX-2 expression by altering NF-kappaB signaling pathway via ERK and p38 MAPK, as well as TPA-induced ODC expression in mouse skin. Thereby, hemin may be an attractive candidate for a chemopreventive agent.     

Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity

The function of the unique axial thiolate ligand of cytochrome P450 has been investigated by mutagenesis of the active-site cysteine with other amino acids in NH(2)-truncated P450s 2B4 and 2E1. The expressed Ser-436 variant of P450 2B4 was highly purified but incurred considerable heme loss. The pyridine hemochrome spectrum of C436S is characteristic of protoporphyrin IX, and the absolute spectra display Soret maxima at 405 nm (ferric), 422 nm (ferrous), and 413 nm (ferrous CO). 2B4:C436S catalyzes the NADPH- and time-dependent formation of H(2)O(2) in the reconstituted enzyme system, with maximal rates at approximately equimolar amounts of P450 reductase and C436S hemeprotein. The 2-electron oxidase activity with saturating reductase is directly proportional to the concentration of 2B4:C436S, and the turnover is 60-70% of that of the wild-type enzyme. In contrast, the C436S variant is devoid of oxygenase activity with typical substrates such as d-benzphetamine, 1-phenylethanol, and 4-fluorophenol, and has only marginal 4-nitrophenol aromatic hydroxylation activity. H(2)O(2)-supported peroxidation of guaiacol and pyrogallol is comparable with 2B4 and mutant C436S and negligible relative to the turnover of peroxidases with these substrates. Neither 2B4 nor 2B4:C436S catalyzes H(2)O(2) decomposition. It is concluded that replacement of active-site Cys-436 by Ser converts P450 2B4 mainly into a 2-electron oxidase.