Iron Protoporphyrin Research Articles

Microburger Biochemistry: Extraction and Spectral Characterization of Myoglyobin from Hamburger

This experiment provides a demonstration of useful biochemical methods at a Basic or Advanced Level, depending upon the available spectrophotometric equipment. The protocol combines protein extraction, ox-i-dation and reduction, and simple spectroscopic analysis, as well as gel filtration chromatography and generation/analysis of spectral scans. Mammalian myoglobin (Mb) is a monomeric O2-binding protein that functions in muscle to store oxygen. The single iron protoporphyrin IX (heme) group is bound to protein by the amino acid Histidine93. The common, stable forms, Met-Mb and Oxy-Mb are studied because in a non-living system, red Oxy-Mb is converted to brown Met-Mb as bound O2 molecule is released. Mb is easily extracted from steak, to illustrate and address why fresh meat is red and aged meat is brown; the protein has unique spectral properties that are diagnostic for characterization of sample identity. After application of heme redox chemical methods, the MetMb or OxyMb samples can be studied spectros...

Resonance Raman spectroscopic characterization of alpha-hydroxyheme and verdoheme complexes of heme oxygenase.

Heme oxygenase (HO) is the microsomal enzyme that catalyzes the oxidative degradation of protoheme (iron protoporphyrin IX) and the generation of carbon monoxide. The enzyme converts protoheme into biliverdin through two known heme derivatives, alpha-hydroxyheme and verdoheme. To gain insight into the degradation mechanisms of the two intermediates, the resonance Raman spectra were observed for alpha-hydroxyheme and verdoheme complexes of HO and compared with those of apomyoglobin (apo-Mb) complexes. The ferrous alpha-hydroxyheme complexed with both HO and apo-Mb shows a resonance Raman spectral pattern similar to that of the protoheme complexes. On the contrary, the ferric alpha-hydroxyheme and ferrous verdoheme complexes of HO and apo-Mb show atypical Raman patterns, which are interpreted as the result of the symmetry lowering of the porphyrin-conjugated pi-electron system. The comparison of the resonance Raman spectra of the verdoheme complexed with HO and apo-Mb with those of the five- and six-coordinate model complexes of verdoheme shows that the ferrous forms of the verdoheme-protein complexes are six-coordinate. The Fe-CO and Fe-CN stretching frequencies of ferrous verdoheme compounds are distinct from those of ferrous heme compounds. It is inferred that the positive charge of the verdoheme ring possesses some of the charge density on the iron atom, causing unique characteristics of the iron ligand stretching vibrations and altered ligand binding properties.

Purification and characterization of two manganese peroxidase isozymes from the white-rot basidiomycete Dichomitus squalens

Two manganese peroxidase isozymes, MnP1 and MnP2, were purified from the extracellular medium of ligninolytic cultures of Dichomitus squalens. The proteins were purified to homogeneity using DEAE-Sepharose chromatography and Mono Q fast protein liquid chromatography. MnP1 and MnP2 have molecular masses of 48 000 and 48 900 Da, as respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both isozymes are glycoproteins and each contains one iron protoporphyrin IX as a prosthetic group. The p I values of MnP1 and MnP2 are 4.15 and 3.90, respectively. N-Terminal amino-acid analysis suggests that these proteins are encoded by distinct genes. The Soret bands of the native ferric enzymes (408 nm and 406 nm, respectively) are shifted to 434 nm in the reduced enzymes and to 422 nm in the reduced-CO complexes. EPR g-values of the native enzymes are essentially identical to those for other MnPs and lignin peroxidases, and they confirm the high-spin state of the iron. The addition of 1 equivalent of H 2O 2 to either of the native ferric isozymes yields spectra which are characteristic of compound I. Successive additions of 1 equivalent of ferrocyanide and 1 equivalent of H 2O 2 to the native enzymes yield spectra which are characteristic of compound II. Both MnP isozymes oxidize Mn 2+ to Mn 3+ in the presence of organic acid chelators. The MnP isozymes are produced by D. squalens only when the cells are grown in the presence of Mn.

Haemin binding as a factor in the virulence of Porphyromonas gingivalis

Haemin (iron protoporphyrin IX) is an essential growth factor for the periodontal pathogen, Porphyromonas gingivalis. Iron protoporphyrin IX (IPP IX) binding to the avirulent P. gingivalis beige variant (W50/BE1) and the black-pigmenting parent wild-type strain W50 was quantified. W50/BE1 grown in a chemostat under haemin excess-bound IPP IX under both oxidising and reducing conditions but with both lower capacity and avidity than either the haemin-limited- and haemin-excess-grown parent strain W50. Rosenthal plots for W50/BE1 indicated cooperative binding. W50/BE1 cells expressed a 32 kDa outer membrane haemin-binding protein when grown under conditions of haemin excess, and this strain might serve as a useful source from which to isolate this protein. The reduced IPP IX binding ability of W50/BE1 may be the rate-limiting factor for haem uptake and explain the reduced virulence and slower rate of pigmentation of this strain.

Haemin binding as a factor in the virulence of Porphyromonas gingivalis.

Haemin (iron protoporphyrin IX) is an essential growth factor for the periodontal pathogen. Porphyromonas gingivalis. Iron protoporphyrin IX (IPP IX) binding to the avirulent P. gingivalis beige variant (W50/BE1) and the black-pigmenting parent wild-type strain W50 was quantified. W50/BE1 grown in a chemostat under haemin excess-bound IPP IX under both oxidising and reducing conditions but with both lower capacity and avidity than either the haemin-limited- and haemin-excess-grown parent strain W50. Rosenthal plots for W50/BE1 indicated cooperative binding. W50/BE1 cells expressed a 32 kDa outer membrane haemin-binding protein when grown under conditions of haemin excess, and this strain might serve as a useful source from which to isolate this protein. The reduced IPP IX binding ability of W50/BE1 may be the rate-limiting factor for haem uptake and explain the reduced virulence and slower rate of pigmentation of this strain.

Electrochemical redox reactions of hemin derivatives having thienylene groups in ion conductive PEO oligomers

Hemin derivative having thienylene groups, hemin thienyl ester (HTE), was synthesized and its electrochemical behavior was analyzed in poly(ethylene oxide) (PEO) oligomers. A cyclic voltammogram of the HTE dissolved in dimethylformamide (DMF) showed redox peaks (E 1 2 = − 0.10 V vs. Ag) based on the heme (iron protoporphyrin IX). The HTE also showed the same redox behavior in PEO 200 (molar mass of 200). Symmetrical redox waves were observed in the cyclic voltammogram of the HTE adsorbed on the indium tin oxide (ITO) coated glass electrode in PEO 200. Its peak separation was about 10 mV and the quantity of electricity was constant and both were independent of the scan rate. These results indicated that HTE molecules were fixed on the ITO electrode as a monolayer. The thienylene group was confirmed to be effective to fix the redox active molecules on the ITO electrode suitable for the electron transfer even in the PEO oligomers.

COMPARISON OF LUMINOL CHEMILUMINESCENCE WITH ATP BIOLUMINESCENCE FOR THE ESTIMATION OF TOTAL BACTERIAL LOAD IN PURE CULTURES

A rapid chemiluminescent assay of total bacterial load that is based on the oxidation of luminol (5‐amino‐2,3‐dihydro‐1,4‐phthalazinedione) as catalyzed by bacterial iron protoporphyrins is described and compared to the ATP bioluminescent assay of microbial biomass. An assay format that elicits linear light output response to a range of analyte concentrations of model compounds such as hematin and various heme‐containing enzymes within the dynamic range of a BioOrbit 1251 luminometer is presented. When the assay was applied to eight pure bacterial cultures, the sensitivity was typically in the range of 104‐105 cfu/ml, and was comparable to that obtained by the ATP assay. Similar levels of sensitivity can be derived from estimates of average values of 2.8 × 10‐18 mole of heme/cfu and 1 × 10‐19 mole of ATP/cfu. The potential of the luminal assay as an alternative rapid test for the estimation of total bacterial count in food and environmental samples is discussed.

Complexity and Diversity of Mammalian Adenylyl Cyclases

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Oxidative Stress in Neurodegenerative Diseases

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Exocytosis: Proteins and Perturbations

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe2+, carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism ...Read More

Structure and Dynamics of CO−Iron(II) Protoporphyrin IX in Dimethyl Sulfoxide

In this report we examine the steady-state optical absorption, steady state and transient vibrational structure, and ligand rebinding kinetics of (CO)FeII protoporphyrin IX ((CO)FeIIPPIX) in dimethyl sulfoxide (DMSO). Steady state optical absorption and resonance Raman spectra of this complex are characteristic of heme iron that is six-coordinate and low-spin. Absorption maxima are observed at 415 nm (Soret), 568 nm (α-band), and 535 nm (β-band), and vibrational bands are observed at 1370 cm-1 (ν4), 1496 cm-1 (ν3), 1551 cm-1 (ν38), 1584 cm-1 (ν2), and 1626 cm-1 (νCC). The transient absorption difference spectrum subsequent to photolysis displays an absorption maximum at 433 nm and a minimum at 414 nm that decays biphasically with pseudo-first-order rate constants of (2.11 ± 0.024) × 106 s-1 and (2.29 ± 0.036) × 102 s-1. The corresponding transient resonance Raman spectrum displays vibrational bands at 1356 cm-1 (ν4), 1470 cm-1 (ν3), 1559 cm-1 (ν2), 1584 cm-1 (ν37), and 1618 cm-1 (νCC). These results are c...

Understanding the structural aspects of neuronal nitric oxide synthase (NOS) using microdissection by molecular cloning techniques: molecular dissection of neuronal NOS.

The neuronal isoform (Type I) of nitric oxide synthase (NOS) requires Ca+2/calmodulin to catalyze the formation of NO● and citrulline from L-arginine (1) and molecular oxygen (2) with reducing equivalents from NADPH. The overall reaction is a five-electron process involving two successive monooxygenation steps with the obligatory formation of N-hydroxy-L-arginine as the oxygenated intermediate. The neuronal NOS shares with the other two known isoforms the unusual property, for a mammalian enzyme, of containing iron protoporphyrin IX (3–6), FAD and FMN (3, 7–9), and tetrahydrobiopterin (9). Bredt, et al. (10) had previously demonstrated the remarkable (58%) sequence similarity of rat brain NOS to rat liver NADPH-cytochrome P450 reductase and determined that the C-terminal 641 amino acids of NOS display consensus regions for both flavin and nucleotide binding. It was reported that carbon monoxide (CO) inhibited both macrophage and neuronal NOS activity (3–6) and these various preparations exhibited reduced-CO difference spectra with absorbance maxima in the region of 445 nm, a property shared with the members of the cytochrome P450 family. However, the low degree of sequence similarity to any of the more than 200 members of this family and other CO-binding, oxygenating heme proteins, such as chloroperoxidase or Bacillus megaterium P450 (BM-3), suggests that the homology may end with the sharing of a cysteine thiolate ligand to the heme iron at the fifth axial position. Recent studies have established that this involves cysteine415 of the neuronal NOS (11, 12), as suggested originally by McMillan, et al. (3), and cysteine184 of the endothelial NOS (13) as determined by site-directed mutagenesis of the holoenzyme (11,13) and the heme domain (12), respectively.

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.

In Situ STM and AFM Study of Protoporphyrin and Iron(III) and Zinc(II) Protoporphyrins Adsorbed on Graphite in Aqueous Solutions

Iron(III) protoporphyrin(IX), zinc(II) protoporphyrin(IX), and protoporphyrin(IX) have been studied on the graphite basal plane in aqueous solutions with both scanning tunneling (STM) and atomic force (AFM) microscopies. Real-time images directly show that the molecules adsorb onto the electrode and condense into monolayer films starting from small islands. In the monolayer films, all three molecules lie flat on the surface and form identical two-dimensional lattices with a = 13.4 ± 0.2 A, b = 12.2 ± 02 A, and γ = 68 ± 2°. The corresponding molecular packing density is 1.1 x 10 -10 mol/cm 2 , which is in excellent agreement with the value determined from cyclic voltammograms. Although the three molecules are nearly identical in their geometrical structures, their internal structures revealed by STM are significantly different. After the monolayer is completed, iron(III) protoporphyrin(IX) forms aggregates with a thickness of ∼8.5 A, while zinc(II) protoporphyrin(IX) and protoporphyrin(IX) do not.

High-Pressure-Assisted Reconstitution of Recombinant Chloroperoxidase

An expression vector containing a T7 promoter and an OmpA signal sequence followed by the DNA sequence of mature chloroperoxidase from the fungus Caldariomyces fumago has been transformed into Escherichia coli. This construct gave high-level expression of apochloroperoxidase when induced with isopropyl thiogalactopyranoside. The nonglycosylated apoenzyme was secreted into periplasmic space. The recombinant apochloroperoxidase was expressed at a level representing about 2% of the total cellular protein. Before conversion to holoenzyme, the apochloroperoxidase was denatured in 8 M urea and partially purified by DEAE chromatography. Maximum yields of holoenzyme were obtained when the denatured apochloroperoxidase, dissolved in a refolding buffer containing iron protoporphyrin IX, calcium ions, and oxidized glutathione, was subjected to high pressure (207 MPa) at -12 degrees C and then allowed to refold at atmospheric pressure and room temperature. The recombinant holoenzyme was characterized by absorption and CD spectroscopy and tested for halogenation and peroxidation activity. The yield of active holochloroperoxidase was about 5% when high-pressure treatment was used as part of the reconstitution process. In the absence of pressure treatment, holoenzyme was formed at about the 1% level. The holochloroperoxidase preparations which resulted from high-pressure treatment showed, upon return to atmospheric pressure, a considerably higher content of native-like secondary structure compared to the nonpressurized preparations. These experiments show that active recombinant chloroperoxidase molecules can be produced, and prove that glycosylation is not a mandatory requirement for chloroperoxidase refolding.

Subunit Dissociation and Unfolding of Macrophage NO Synthase: Relationship between Enzyme Structure, Prosthetic Group Binding, and Catalytic Function

Macrophage NO synthase is a homodimer of 130 kDa subunits. Each subunit contains an oxygenase domain that binds iron protoporphyrin IX (heme) and tetrahydrobiopterin (H4biopterin) and a reductase domain that binds FAD, FMN, and calmodulin (CaM) [Ghosh & Stuehr (1995) Biochemistry 34, 801-807]. We have studied the dissociation and unfolding reactions of dimeric iNOS in urea to learn how enzyme structure relates to catalysis and prosthetic group binding. The iNOS dimer dissociated between 0 and 2.5 M urea, and the subunits partially unfolded at 2.5 M urea and above. Dimer dissociation was accompanied by loss of NO synthesis activity and release of bound H4biopterin from the protein. However, the dissociated subunits maintained their cytochrome c and ferricyanide reductase activities and retained near stoichiometric quantities of bound heme. The subunit unfolding transition was accompanied by loss of reductase activities and partial loss of bound heme but retention of bound flavins and CaM. The heme iron in the dissociated subunits remained coordinated through axial cysteine thiolate ligation. Kinetic analysis of dimer dissociation showed that loss of NO synthesis correlated with a loss of heme Soret absorbance at 398 nm and an appearance of absorbance bands at 377 and 460 nm, which were attributed to DTT coordination to the sixth position of the heme iron to form a mixed bisthiolate complex. Subunits could reassociate into a dimer when incubated with L-arginine and H4biopterin. Dimer formation correlated with proportional recoveries of NO synthesis and heme Soret absorbance at 398 nm. Thus, dimeric iNOS undergoes separate dissociation and unfolding transitions in urea, and each transition is accompanied by a loss of a specific catalytic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and Characterization of the Mycobacterium smegmatis Catalase-Peroxidase Involved in Isoniazid Activation

The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.

Kinetics of Congo-red Binding by Haemin-limited and Haemin-excess Cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.

Molecular mechanisms and therapeutic approaches to the treatment of African trypanosomiasis.

There are only a handful of drugs available today for treating African trypanosomiasis, most of which were discovered more than forty years ago. These drugs are plagued by various problems, ranging from oral in-absorption, acute toxicities, short durations of action, and low efficacies to the emergence of trypanosomal resistance. Mechanisms of antitrypanosomal action of these drugs are mostly unknown, except for eflornithine, which is a suicide inhibitor of ornithine decarboxylase. On the other hand, the African trypanosomes are among the most extensively studied parasitic protozoa to date. Many of their intriguing biological features have been well documented and can be viewed as attractive targets for antitrypanosomal chemotherapy. These features include the glycosomal functions and protein import, the trans-splicing of mRNAs, the machineries for controlled protein degradations, the polyamine metabolism, the trypanothione metabolism, the purine salvage enzymes, and the glycolipid anchor for the surface glycoproteins.

Congo red binding by Porphyromonas gingivalis is mediated by a 66 kDa outer-membrane protein

Congo red was bound from solution by strains of Porphyromonas gingivalis including W50, HG189, HG184, NCTC 11834, Bg 381, WPH35, the slower brown pigmenting colonial variant W50/BR1, and the avirulent mutant W50/BE1, and by Porphyromonas endodontalis HG370 and Porphyromonas asaccharolytica B537. SDS-PAGE of whole cells of all species examined displayed a 66 kDa Congo-red-binding component which was also detected in the outer membranes of P. gingivalis W50 grown in the chemostat under both haemin limitation and haemin excess, and which corresponded to a Coomassie-blue-stained band of the same mobility. Pretreatment of haemin-excess batch-grown cells of P. gingivalis W50 with polymyxin B, which binds to lipid A, did not inhibit binding, whilst binding was enhanced in the presence of 2 M ammonium sulphate, suggesting the involvement of non-specific hydrophobic interactions. Binding was also reduced by pretreatment with trypsin and papain, and by 8-anilino-1-naphthalenesulphonic acid, which binds to hydrophobic amino acids. The 66 kDa binding component was sensitive to proteinase K digestion, and loss of Congo red staining of this band correlated with the quantitative reduction in Congo red binding by whole cells. These data, and our previous work, show that Congo red and iron protoporphyrin IX (haemin) are bound to different outer-membrane components, and that Congo red binding may be of little value as a marker to detect virulent strains of P. gingivalis or those expressing haemin-binding proteins.