Iron Protoporphyrin Research Articles

Inhibition of prostaglandin H2 synthases by salicylate is dependent on the oxidative state of the enzymes.

At antipyretic and analgesic doses, salicylate has no antiplatelet or anti-inflammatory effects, unlike typical inhibitors of the prostaglandin H synthases (PGHSs). We demonstrated that salicylate inhibits PGHS-1 and -2 with a potency inversely related to ambient hydroperoxide concentrations. Salicylate inhibition of PGHS-1 was prevented by 12-hydroperoxyeicosatetraenoic acid (12-HPETE). Increasing the production of prostaglandin G2 (the peroxide product of PGHS-cyclooxygenase activity) by elevating the concentration of either enzyme or substrate reversed inhibition. Using analogs of benzoic acid differing only at the hydroxyl position, we revealed the importance of this moiety to salicylate's inhibitory mechanism. Unlike typical phenolic inhibitors, e.g., acetaminophen, salicylate was ineffective as a reducing cosubstrate for PGHS-peroxidase activity, implicating the cyclooxygenase site as its putative target. PGHS-cyclooxygenase activity depends upon the oxidation of an active site tyrosine by electron transfer to the oxidized ferriprotoporphyrin of the peroxidase. The PGHS-1 apoenzyme reconstituted with manganese protoporphyrin instead of iron protoporphyrin has very little peroxidase activity. 12-HPETE does not prevent the inhibition of Mn-PGHS-1 by salicylate, indicating that reversal of salicylate inhibition by hydroperoxides depends upon electron transfer between the cyclooxygenase and peroxidase active sites. These results are consistent with an inhibitory action of salicylate at the PGHS-cyclooxygenase site that is dependent on the PGHS-peroxidase activity.

Cloning and expression of a Porphyromonas gingivalis gene for protoporphyrinogen oxidase by complementation of a hemG mutant of Escherichia coli.

Porphyromonas gingivalis, a bacterium implicated in periodontal pathogenesis, has a growth requirement for iron protoporphyrin IX. By complementation with a P. gingivalis 381 chromosomal DNA library, we were able to isolate a clone that enhanced the poor growth of a hemG mutant of Escherichia coli. The DNA sequence analysis of this clone revealed three open reading frames (ORFs). ORF3 encoded a protein of 466 amino acids with a calculated molecular weight of 51 695 Da. The deduced amino acid sequence of the ORF3 gene had significant similarity to sequences of protoporphyrinogen oxidase (PPO) from Myxococcus xanthus (30% identical residues). When the ORF3 gene was overexpressed in E. coli, the extract had much higher PPO activity than a control extract, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Thus, ORF3 was named PgHemG. Furthermore, several porphyrin-related genes, including hemD, hemN and hemH, were identified in the data bases on the websites available on-line. We postulated that a porphyrin biosynthetic pathway to heme from preuroporphyrin may be conserved in P. gingivalis.

Truncated hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002: evidence for hexacoordination and covalent adduct formation in the ferric recombinant protein.

The glbN gene for the hemoglobin of Synechoccocus sp. PCC 7002, a cyanobacterium incapable of nitrogen fixation, was cloned and overexpressed in Escherichia coli. The 123-residue protein was purified from inclusion bodies and reconstituted with iron protoporphyrin IX to obtain the ferric form of the holoprotein. Mass spectrometric analysis confirmed the identity of the polypeptide. NMR and optical data demonstrated that the protein so prepared contained a hexacoordinate heme group, as observed in the related globin from Synechocystis sp. PCC 6803 [Scott, N. L., and Lecomte, J. T. J. (2000) Protein Sci. 9, 587-597]. The data were consistent with a similar bis-histidine coordination scheme involving His46 (E10) on the distal side and His70 (F8) on the proximal side. Several aromatic residues were identified in the vicinity of the heme and were used to establish the orientation of the prosthetic group in the polypeptide matrix. In this protein, as in that from Synechocystis sp. PCC 6803, there was a marked preference for the heme orientation in which pyrroles C and D contact the C-E corner of the protein. Both hemoglobins were found capable of forming a product in which the heme is cross-linked to the polypeptide through modification of a vinyl group.

Interactions of Porphyromonas gingivalis with oxyhaemoglobin and deoxyhaemoglobin

When grown on blood-containing solid media, the anaerobic periodontal pathogen Porphyromonas gingivalis produces a haem pigment, the major component of which is the μ-oxo bishaem of iron protoporphyrin IX [Smalley, Silver, Marsh and Birss (1998) Biochem. J. 331, 681–685]. In this study, μ-oxo bishaem generation by P. gingivalis from oxy- and deoxyhaemoglobin was examined. Bacterial cells were shown to convert oxyhaemoglobin into methaemoglobin, which was degraded progressively, generating a mixture of both monomeric and μ-oxo dimeric iron protoporphyrin IX. The rate of methaemoglobin formation was accelerated in the presence of bacterial cells, but was inhibited by N-ethylmaleimide and tosyl-lysylchloromethylketone. Interaction of cells with deoxyhaemoglobin resulted in formation of an iron(III) haem species (Soret λmax, 393nm), identified as pure μ-oxo bishaem.

Interactions of Porphyromonas gingivalis with oxyhaemoglobin and deoxyhaemoglobin.

When grown on blood-containing solid media, the anaerobic periodontal pathogen Porphyromonas gingivalis produces a haem pigment, the major component of which is the mu-oxo bishaem of iron protoporphyrin IX [Smalley, Silver, Marsh and Birss (1998) Biochem. J. 331, 681-685]. In this study, mu-oxo bishaem generation by P. gingivalis from oxy- and deoxyhaemoglobin was examined. Bacterial cells were shown to convert oxyhaemoglobin into methaemoglobin, which was degraded progressively, generating a mixture of both monomeric and mu-oxo dimeric iron protoporphyrin IX. The rate of methaemoglobin formation was accelerated in the presence of bacterial cells, but was inhibited by N-ethylmaleimide and tosyl-lysylchloromethylketone. Interaction of cells with deoxyhaemoglobin resulted in formation of an iron(III) haem species (Soret gamma(max), 393 nm), identified as pure mu-oxo bishaem.

Environment effects on chemical reactivity of heme proteins

AbstractHeme proteins are involved in a variety of physiological processes, such as O2 transport, electron transfer, sensing of O2 or CO, and catalysis of redox reactions. Despite the differences in biologic function, all these proteins have iron protoporphyrin IX (heme b) as the active site. The amino acids surrounding the active site are responsible for the specific reactivity of each protein. We analyzed the environment effects on binding of small ligands such as O2 and NO to several heme proteins using density functional theory (DFT) calculations of model systems including selected amino acid residues, and also DFT calculations of the active site coupled to an electrostatic representation of the rest of the protein. Specifically, we considered the following problems: (1) the mechanisms underlying inactivation by nitric oxide of cytochrome P450; (2) O2 affinity of human and Ascaris hemoglobin and the role of oxygen hydrogen bonding to the distal amino acids; (3) the influence of the amino acid residues surrounding the proximal histidine in the Fehistidine bond cleavage upon binding of NO in FixL, horseradish peroxidase, and human hemoglobin. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002

Designer haem proteins: What can we learn from protein engineering?

AbstractIron protoporphyrin(IX) is one of the most versatile and widespread pieces of catalytic machinery known in biology and is a key component of a multitude of proteins and enzymes. One of most challenging questions in this area has been to identify and understand the relationships that exist between different classes of haem proteins and to use protein engineering methods to rationalize the mechanisms by which the protein structure controls the specific chemical reactivity of the haem group. The application of this approach to the haem enzyme ascorbate peroxidase and the haem protein leghaemoglobin is discussed. © 2002 Wiley Periodicals, Inc. Heteroatom Chem 13:501–505, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hc.10094

Nitric oxide synthases: structure, function and inhibition.

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

Nitric oxide synthases: structure, function and inhibition

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, l-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

Detection of heme-binding proteins in epidemic strains of Burkholderia cepacia.

A panel of 30 previously characterized strains representing five genomovars from the Burkholderia cepacia complex (E. Mahenthiralingam, T. Coenye, J. W. Chung, D. P. Speert, J. R. W. Govan, P. Taylor, and P. Vandamme, J. Clin. Microbiol. 38:910--913, 2000) were examined for their iron protoporphyrin IX-binding ability. These included B. cepacia genomovars I and III and B. stabilis (formerly B. cepacia genomovar IV), B. multivorans (formerly B. cepacia genomovar II), and B. vietnamiensis (formerly B. cepacia genomovar V). Cells were exposed to micro-oxo bisheme of iron protoporphyrin IX (micro-oxo dimers) and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing, nondenaturing conditions for the presence of heme-binding proteins using tetramethylbenzidine-H(2)O(2) staining. Seven of the 30 strains, each belonging to B. cepacia genomovar III and designated epidemic (in possessing the B. cepacia epidemic strain marker), expressed a 96- to 100-kDa heme-binding protein which was located in the outer membrane. The heme-binding protein of B. cepacia genomovar III epidemic strain C5424 bound iron(III) protoporphyrin IX in both the monomeric and micro-oxo bisheme forms. Cells of all strains grown on Columbia agar bound iron protoporphyrin IX in the micro-oxo bisheme (dimeric) form. There were no statistical differences between the five genomovars, or those possessing the heme-binding protein, in their micro-oxo bisheme-binding ability. Possession of the outer membrane heme-binding protein may be a pathogenicity trait in enabling the bacterium to withstand oxidative stresses in inflammatory exudates in the lung and may aid identification of invasive epidemic strains of B. cepacia.

Photoreduction of iron protoporphyrin IX chloride in non‐ionic triton X‐100 micelle studied by electronic absorption and resonance Raman spectroscopy

AbstractResonance Raman and electronic absorption studies of iron protoporphyrin IX chloride (hemin) in non‐ionic Triton X‐100 micelle in the absence and presence of hindered imidazole (2‐methylimidazole and 1,2‐dimethylimidazole) and unhindered imidazole under various experimental conditions are reported. Hemin undergoes photoreduction at the metal center, both in the absence and presence of hindered imidazole, in anaerobic, alkaline and neutral pH conditions on photoexcitation by laser radiation at 441.6 and 457.9 nm. It is inferred from this study that only the monomer hemin encapsulated within the micelle under the alkaline pH conditions is photoreducible. The photoreduction of hemin in this micelle occurs from an electron transfer as a result of dissociation of coordinated hydroxyl ion to the iron atom in the photoexcited state, which may also involve the OH→Fe charge transfer transition around 360 nm. Copyright © 2001 John Wiley & Sons, Ltd.

Induced Heme Oxygenase-1 Upregulation Protects Pancreatic Beta Cells from Apoptosis In Vitro.

INTRODUCTION. Transplantation of islets of Langerhans represents a viable therapeutic approach for the treatment of Type I diabetes mellitus (1). Unfortunately, transplanted islets are susceptible to allogeneic recognition and rejection, recurrence of autoimmunity and destruction by local inflammation at the site of implant. The latter phenomenon is characterized by microenvironment activation with production of proinflammatory cytokines (IL-1, IFN-γ, TNF-α) and oxygen radicals that might result in functional impairment and death of islet cells, and also contribute to amplifying the subsequent specific immune response (2,3). Islet β−cells are exquisitely susceptible to oxidative stress because of their insufficient anti-oxidant pool. Induction of islet cell protection against inflammation could therefore represent a viable strategy to improve overall graft fate. Heme oxygenase 1 (HO-1) has been described as an inducible stress protein capable of cytoprotection via radical scavenging and apoptosis prevention (4). The purpose of the present study was to analyze whether upregulation of HO-1 in a beta cell line (β-TC3) and in freshly isolated murine islets could result in protection from apoptosis. METHODS AND RESULTS. HO-1 upregulation was reproducibly induced, in a dose dependent fashion, by incubating the beta cell line and isolated islets for 24 hours in the presence of iron protoporphyrin (FePP), as assessed by western blot and quantitative RT-PCR analysis. A 24-hr incubation in the presence or absence of FePP was performed in both BetaTC3 cells and islets before induction of apoptosis. Subsequently, β-TC3 cells were incubated for 24-hrs in the presence of IL-1α (10U/ml) and IFN-γ (100U/ml) to induce the expression of Fas on their surface. A pro-apoptotic stimulus was then delivered via engagement of Fas by an agonistic anti-Fas antibody, followed by addition of protein G to induce cross-linking, and cells were cultured for 48-hrs before assay. In freshly isolated murine islets, the pro-apoptotic signal was delivered by addition of TNF-α (5000U/ml) and cyclohexamide (CHX, 50 µg/ml) for additional 48 hours. Control islets were similarly treated in the presence or absence of FePP, without TNF-α and CHX. At the end of culture, β-TC3 and islet cell suspension were obtained by trypsinization, stained with propidium iodide, and analyzed by flow cytometry on a linear scale, considering apoptotic the cells within the distinct sub-G1 peak, in which DNA condensation and fragmentation results in decreased staining. Analysis of apoptosis in the β-TC3 showed that the rate of apoptotic cells was inversely correlated to the concentration of FePP in the media, strongly suggesting a dose

Heme redox potential control in de novo designed four-alpha-helix bundle proteins.

The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [¿CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()¿(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.

Temperature elevation regulates iron protoporphyrin IX and hemoglobin binding by Porphyromonas gingivalis.

Porphyromonas gingivalis, an obligate anerobe with a growth requirement for iron protoporphyrin IX (FePPIX), is exposed to increased temperatures in the inflamed periodontal pocket. In this study, P. gingivalis was grown in a chemostat at 37 degrees C (control), 39 degrees C, and 41 degrees C, and examined for hemagglutinating (HA) activity, hemoglobin binding and degrading activity, and iron protoporphyrin IX binding. HA activity decreased in cells as the growth temperature increased. Binding of mu-oxo bishaem (dimeric haem), and Fe(II)- and Fe(III)-monomeric forms was increased in 39 degrees C-grown cells but decreased in 41 degrees C-grown cells compared with controls. Cellular hemoglobin binding and degradation decreased with increased growth temperature. The decrease in cellular hemagglutination and hemoglobin degradation occurring with increased growth temperature would limit the potential overproduction of toxic monomeric haem molecules. The increased binding of mu-oxo bishaem and monomeric forms of FePPIX at 39 degrees C may reflect a defense strategy against reactive oxidants and a mechanism of dampening down the inflammatory response to maintain an ecological balance.

Studies on Pseudomonas aeruginosa cd1 nitrite reductase: The association and dissociation reactions of the d1‐heme

AbstractThe dissociation and association reactions of the d1‐heme, the prosthetic group characteristic of cd1 nitrite reductases (NiRs), have been investigated to assess the stability of the native enzyme. At pH 5.0 and 37 °C, the rate constant for the dissociation of the ferric d1‐heme from native NiR purified from Pseudomonas aeruginosa is 4.7 ± 1.4 × 10−4 s−1. However, when the d1‐heme is in the ferrous state no dissociation is observed, consistent with the shortening and strengthening of the proximal bond upon reduction of the iron. Recombinant wild‐type protein and two single point mutants (Y10N and Y10F), which are expressed as semi‐apo proteins and were reconstituted with synthetic d1‐heme, display the same slow dissociation rate as the native enzyme. Therefore the stability of the d1‐heme bound in the crevice provided by the eight‐bladed ß propeller domain is not altered by the act of reconstitution or by these two point mutations. The association reaction between the ferric d1‐heme and semi‐apo NiR is second‐order and governed by an apparent rate constant of 3.3 × 106 M−1 s−1 at neutral pH and 25 °C. Interestingly, the combination rate constant is an order of magnitude slower than that reported for iron protoporphyrin IX and apomyoglobins or apohemoglobins. This difference appears to be a property of the d1‐heme and not of the protein since association rate constants of CO‐protoheme‐Fe(II) and dicyanoprotoheme‐Fe(III) with semi‐apo NiR are 5 × 107 M−1 s−1 and 6 × 107 M−1 s−1, respectively. These results are discussed with reference to the structure of the d1‐heme binding site, as inferred from the known 3D structure of P. aeruginosa NiR.

Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical.

Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.

Electrochemistry of Self-Assembled Monolayers of Iron Protoporphyrin IX Attached to Modified Gold Electrodes through Thioether Linkage

Iron(III) protoporphyrin IX (Fe(III)PP) and iron(III) hematoporphyrin (Fe(III)HP) were selectively and covalently attached to dimercaptoalkane-modified gold electrodes. Reaction of their vinyl or hydroxyethyl groups with the surface-immobilized thiols produced thioether linkages, reminiscent of the heme macrocycle attachment in c-type cytochromes. Cyclic voltammetry revealed reversible electrochemistry of self-assembled monolayers (SAMs) of FePPs and FeHPs on the thiol-modified gold substrates. The surface coverage estimated from the charges transferred corresponds to 30% of a monolayer. The heterogeneous rate constant of electron transfer between the Fe(III)PPs and the gold substrate decreases exponentially with the length of the spacer, demonstrating a value of 1.0 Å-1 for the tunneling length coefficient, β. At pH 8, a linear increase of the formal redox potential (E°‘) with the length of the linker was also observed. This suggests that in the film, there is a close contact between the porphyrins and the alkane SAM: the E°‘ is affected by the drop of the electrostatic potential from the electrode to the surface of the alkane SAM, and also that there is a strong ion pairing of the Fe(III)PPs in the film with the anions of the solution. The E°‘ of Fe(III)PPs in the SAM shows a strong and complex dependency on the pH of the solution, explained by variations in the coordination of the iron, involving hydroxyl ions, water, and eventually dioxygen molecules. Interactions of the iron with either functional groups present at the surface of the substrate or with the propionate groups attached to the porphyrin ring, do not appear to be involved in the electron−proton transfer coupling mechanisms.

The periodontal pathogenPorphyromonas gingivalisharnesses the chemistry of the μ-oxo bishaem of iron protoporphyrin IX to protect against hydrogen peroxide

The major haem component in the black pigment of Porphyromonas gingivalis is the mu-oxo bishaem of iron protoporphyrin IX and formation and cell-surface binding of this haem species is proposed as an extracellular buffer against reactive oxidants [Smalley, J.W. et al. (1998) Biochem. J. 331, 681-685]. P. gingivalis cells grown in the presence of the mu-oxo bishaem were protected against H(2)O(2) compared to control cells grown without it. When added to the growth medium, soluble mu-oxo bishaem inactivated H(2)O(2) and supported cell growth. Cells carrying a surface layer of mu-oxo bishaem were less susceptible to peroxidation by H(2)O(2). Cell-surface haems were slowly destroyed during reaction with H(2)O(2). Binding of mu-oxo bishaem by P. gingivalis may aid survival during neutrophil attack through inactivation of hydrogen peroxide.

The periodontal pathogen Porphyromonas gingivalis harnesses the chemistry of the μ-oxo bishaem of iron protoporphyrin IX to protect against hydrogen peroxide

The major haem component in the black pigment of Porphyromonas gingivalis is the μ-oxo bishaem of iron protoporphyrin IX and formation and cell-surface binding of this haem species is proposed as an extracellular buffer against reactive oxidants [Smalley, J.W. et al. (1998) Biochem. J. 331, 681–685]. P. gingivalis cells grown in the presence of the μ-oxo bishaem were protected against H 2O 2 compared to control cells grown without it. When added to the growth medium, soluble μ-oxo bishaem inactivated H 2O 2 and supported cell growth. Cells carrying a surface layer of μ-oxo bishaem were less susceptible to peroxidation by H 2O 2. Cell-surface haems were slowly destroyed during reaction with H 2O 2. Binding of μ-oxo bishaem by P. gingivalis may aid survival during neutrophil attack through inactivation of hydrogen peroxide.

Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival.

Decapeptides derived from human HLA class I sequences have been shown to prolong allograft survival. The mechanism of action of these peptides has been uncertain, because they act in an MHC unrestricted manner. Recently, it was found that these peptides bind heme oxygenase 1 (HO-1). In the present study, we sought to determine whether local delivery of these peptides through gene transfer could extend allograft survival, and to explore the underlying mechanisms. C57BL/6 neonatal hearts were transplanted to CBA/J recipients and the peptide, or plasmid DNA encoding the peptide, was injected directly into the allograft at the time of the transplant. Direct injection of 1 microg of the B2702 peptide into the allograft did not prolong survival (13.3+/-0.8 vs. 13.4+/-0.8 days for untreated controls), but injection of 400 microg of peptide did extend survival (22.0+/-0.6). Injection of plasmid DNA encoding the B2702 peptide was superior to peptide delivery, extending graft survival to 30.8+/-1.5 days. Similar results were obtained using another plasmid encoding the rationally designed peptide BC1 (28.5+/-1.7), whereas no significant prolongation was observed using a plasmid encoding the control peptide B2705 (16.5+/-1.0). To explore the hypothesis that these peptides exert their immunosuppressive effect by altering HO-1 activity, animals were treated with iron protoporphyrin, an inducer of HO-1 activity, or tin protoporphyrin, an inhibitor of HO-1. Treatment with iron protoporphyrin alone extended graft survival (24.5+/-1.6) and did not alter the benefit in survival seen with BC1 gene transfer (28.0+/-0.8). In contrast, treatment with tin protoporphyrin abolished the benefit of BC1 gene transfer (17.0+/-0.6). These results demonstrate that plasmid mediated gene transfer is an effective means for delivering immunosuppressive peptides to extend allograft survival. The experiments suggest that these peptides may act by increasing HO-1 activity and support a role for HO-1 in immune regulation and allograft survival.