The one-electron oxidation of porphyrins to porphyrin pi-cation radicals by peroxidases: an electron spin resonance investigation

For the first time, the enzymatic one-electron oxidation of several naturally occurring and synthetic water-soluble porphyrins by peroxidases was investigated by ESR and optical spectroscopy. The ESR spectra of the free radical metabolites of the porphyrins were singlets (g = 2.0024, delta H = 2-3 G), which we assigned to their respective porphyrin pi-cation free radicals. Several porphyrins were investigated and ranked by the intensity of their ESR spectra (coproporphyrin III greater than coproporphyrin I greater than deuteroporphyrin IX greater than mesoporphyrin IX greater than Photofrin II greater than protoporphyrin IX greater than uroporphyrin I greater than uroporphyrin III greater than hematoporphyrin IX). The porphyrins were oxidized by several peroxidases (horseradish peroxidase, lactoperoxidase, and myeloperoxidase), yielding the same type of ESR spectra. From these results, we conclude that porphyrins are substrates for peroxidases. The changes in the visible absorbance spectra of the porphyrins during enzymatic oxidation were monitored. The two-electron oxidation product, which was assigned to the dihydroxyporphyrin, was detected as an intermediate of the oxidation process. The optical spectrum of the porphyrin pi-cation free radical was not detected, probably due to its low steady-state concentration.     

Regulation of soluble guanylate cyclase activity by porphyrins and metalloporphyrins

Alterations of the chemical structure of protoporphyrin IX markedly altered the activation of soluble guanylate cyclase purified from bovine lung. Hydrophobic side chains at positions 2 and 4 and vicinal propionic acid residues at positions 6 and 7 of the porphyrin ring (protoporphyrin IX, mesoporphyrin IX) were essential for maximal enzyme activation (Ka = 7-8 nM; Vmax = 6-8 mumol of cGMP/min/mg). Substitution of hydrophobic with polar groups (hematoporphyrin IX, coproporphyrin III), or with hydrogen atoms ( deuteroporphyrin IX), and methylation of propionate residues resulted in decreased enzyme stimulation. Stimulatory porphyrins increased the Vmax and the apparent affinities of enzyme for MgGTP and uncomplexed Mg2+. An open central core in the porphyrin ring was essential for enzyme activation. The pyrrolic nitrogen adduct, N-phenylprotoporphyrin IX, was inhibitory and competitive with protoporphyrin IX (KI = 73 nM). Similarly, metalloporphyrins inhibited enzymatic activity and ferro-protoporphyrin IX (KI = 350 nM), zinc-protoporphyrin IX (KI = 50 nM) and manganese-protoporphyrin IX (KI = 9 nM) were competitive with protoporphyrin IX. Inhibitory porphyrins and metalloporphyrins also prevented enzyme activation by S-nitroso-N- acetylpenicillamine and NO. Guanylate cyclase reconstituted with such porphyrins required higher concentrations of protoporphyrin IX for further activation and were not activated by NO. Thus, porphyrins, metalloporphyrins, and NO appeared to interact at a common binding site on guanylate cyclase. This common site is likely that which normally binds heme and, therefore, NO-heme when the heme-containing enzyme is exposed to NO. Thus, NO and nitroso compounds may react with enzyme-bound heme to generate a modified porphyrin which structurally resembles protoporphyrin IX in its interaction with guanylate cyclase.     

On the use of iron octa-alkylporphyrins as models for protoporphyrin IX-containing heme systems in studies employing magnetic circular dichroism spectroscopy.

The magnetic circular dichroism (MCD) properties of numerous oxidation and ligation state derivatives of myoglobin and horseradish peroxidase reconstituted with an iron octa-alkylporphyrin (mesoheme IX) have been investigated in order to establish the utility of such porphyrins as models for protoporphyrin IX-containing systems. The MCD spectra of the mesoheme-reconstituted proteins are blue-shifted (4-12 nm) and are somewhat more intense (1.5-2.5 fold) when compared to the spectra of analogous derivatives of native myoglobin and horseradish peroxidase. However, the spectral band patterns of the mesoheme-reconstituted proteins closely resemble those of the native proteins in essentially all cases. These data demonstrate that octa-alkylporphyrins can be productively used as models for protoporphyrin IX in studies of heme proteins with MCD spectroscopy.     

Inhibitory effect of porphyrins on the proliferation of mouse spleen lymphocytes in vitro

The influence of various porphyrins (deuteroporphyrin IX, mesoporphyrin IX, protoporphyrin IX, hematoporphyrin) and two related compounds (hemin, biliverdin) on the spontaneous proliferation of mouse spleen lymphocytes has been estimated in vitro by the 3H-thymidine uptake assay. It has been found that porphyrins (endogenous ligands for the mitochondrial benzodiazepine receptor) produce a concentration-dependent inhibition of 3H-thymidine incorporation into the DNA of these cells. Metalloporphyrin-hemin has been observed to evoke a weak inhibitory effect, in a high concentration (10(-4)M), whereas biliverdin, a porphyrins degradation product, was inactive in the same experimental conditions. Those findings indicate that endogenous porphyrins, presumably acting through the mitochondrial benzodiazepine receptor, could regulate the proliferation of mouse spleen lymphocytes in vitro.     

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.     

Tetrapyrrole utilization by Bacteroids ruminocola.

Reduced versus oxidized difference spectra of whole cells and pyridine hemochromogens of heme-requiring isolates of Bacteroides ruminicola are altered when deuteroporphyrin or mesoporphyrin replaces protoheme as a growth factor. During growth in the presence of either deuteroporphyrin or mesoporphyrin, whole cells exhibit peaks at 545 t547, 515 to 518, and 412 to 413 nm. Pyridine hemochromogen spectra confirm the presence of meso -or deuteroheme in cells grown in the presence of meso- or deuteroporphyrin. No evidence was found for the conversion of either meso- or deuteroporphyrin to protoheme. Cells grown in the presence of the manganese of magnesium chelates of protoheme form iron-containing hemes. Neither spontaneous decomposition of noniron metalloporphyrin chelates nor spontaneous formation of hemes from Fe2+ and metal-free porphyrins was detected. Protoheme-synthesizing isolates of B. ruminicola fail to use preformed metal-free porphyrins, but form both protoheme- and deuteroheme-containing cytochromes when grown in the presence of manganese deuteroheme. Versatility in tetrapyrrole utilization by B. ruminicola appears to reflect the ability of the organism to mediate the removal of nonferrous ions and to insert Fe2+ into the tetrapyrrole nucleus. The orgamism also forms functional b-type cytochromes with prosthetic groups other than protoheme.     

Glutathione-dependent oxidative modification of protoporphyrin and other dicarboxylic porphyrins by mammalian and plant peroxidases.

Protoporphyrin, an intermediate in heme and chlorophyll biosynthesis, can accumulate in human and plant tissues under certain pathological conditions and is a photosensitizer used in cancer phototherapy. We previously showed that protoporphyrin and the related non-natural dicarboxylic porphyrin deuteroporphyrin are rapidly oxidized by horseradish peroxidase in the presence of some thiols, especially glutathione. This study reports that bovine lactoperoxidase, but not leucocyte myeloperoxidase, can also catalyze this reaction and that Tween and ascorbic acid are inhibitors. Exogenous hydrogen peroxide is not required and cannot replace glutathione. Deuteroporphyrin was oxidized to a unique green chlorin product with two oxygen functions added directly to the characteristic reduced pyrrole ring of the chlorin. Spectroscopic and chromatographic results suggest that protoporphyrin was oxidized not to a green chlorin, but to a much more polar red porphyrin modified by oxidative addition to the two vinyl side chains. Two related nonnatural dicarboxylic porphyrins, with ethyl or hydroxyethyl instead of vinyl side chains, are not substrates or products for this enzymatic conversion.     

Sensitized photooxidation of low spin horseradish peroxidase.

Horseradish peroxidase differs from most enzymes in that it is almost completely resistant to photodynamic action due to the paramagnetic ferric ion in the prosthetic group, heme. Chelation of horseradish peroxidase at the sixth coordination position of the iron with a cyanide or hydroxyl group converts it to a low spin diamagnetic state. Upon illumination with visible light with eosin Y, flavin mononucleotide or methylene blue as sensitizer, the low spin enzyme lost both peroxidative and oxidative activities with the same quantum yields. Several amino acid residues, including one histidine and one tyrosine were destroyed in the low spin enzyme after 60 min of illumination with eosin Y as sensitizer.     

Influence of heme-surrounding amino acid residues on the manganese (V)-nitrido bond in manganese-substituted hemoproteins: resonance Raman evidence for porphyrin core expansion and reduction of the manganese(V)-nitrido stretching force constant

Nitridomanganese(V) protoporphyrin IX was prepared by hypochlorite oxidation of the corresponding manganese(III) protoporphyrin IX derivative in the presence of ammonium ion and by photolysis of the corresponding azidomanganese(III) complex. Myoglobin and horseradish peroxidase containing this novel protoporphyrin derivative were prepared for the first time. These remarkably stable species were examined by electronic absorption, electron paramagnetic resonance, and resonance Raman spectroscopies. The MnV-N stretching modes of the nitridomanganese(V)-substituted myoglobin and horseradish peroxidase were observed at 1010 and 1003 cm-1, respectively, by resonance Raman spectroscopy, while the MnV-N stretching frequency for nitridomanganese(V) protoporphyrin IX in 0.1 N aqueous NaOH was found at 1046 cm-1. The equilibrium dissociation energies of MnV-N bonds in these complexes were estimated from vibrational overtone spacings by introducing the Morse potential energy function, were found to be around 4.5 eV, and seemed independent of the surroundings of the manganese porphyrin, although its force constant decreased from 7.3 to 6.7 mdyn/A upon incorporation into apoprotein. The porphyrin ring modes of these nitridomanganese(V) derivatives were influenced greatly upon incorporation into apoproteins, suggestive of the occurrence of porphyrin core expansion. Upon this core expansion the MnV center moves into the mean plane of porphyrin plane, but the access of nitrido (N) toward MnV is restricted due to a steric hindrance from porphyrin pyrrole nitrogens. The resulting stretched MnV-N bond might cause lowering of the MnV-N stretching frequency upon incorporation into apoprotein.     

Equilibrium and kinetic studies of the aggregation of porphyrins in aqueous solution.

An investigation of the behavior of protoporphyrin IX, deuteroporphyrin IX, haematoporphyrin IX and coproporphyrin III in aqueous solution revealed extensive and complex aggregation processes. Protoporphyrin appears to be highly aggregated under all conditions studied. At concentrations below 4 muM, aggregation of deutero-, haemato- and coproporphyrin is probably restricted to dimerization. At approx. 4muM each of these three porphyrins exhibits sharp changes in spectra consistent with a "micellization" process to form large aggregates of unknown size. This critical concentration increases with increasing temperature and pH, but is not very sensitive to variation in ionic strength. Temperature-jump kinetic studies on deuteroporphyrin also imply an initial dimerization process, the rate constants for which are comparable with those for various synthetic porphyrins, followed by a further extensive aggragation. The ability of a particular porphyrin to dimerize appears to parallel that of the corresponding iron(III) complexes (ferrihaems), although it is thought that ferrihaems do not exhibit further aggregation under these conditions.     

Spectrofluorimetric study of porphyrin incorporation into membrane models--evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK approximately equal to 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K approximately equal to 2 X 10(5) and K approximately equal to 6 X 10(6) M-1 (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.     

Photodynamic effect of deuteroporphyrin IX and hematoporphyrin derivatives on single neuron

The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

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.     

Spectrofluorimetric study of porphyrin incorporation into membrane models - evidence for pH effects

The effects of hydrophobicity and charges of dicarboxylic porphyrins upon their interactions with membrane model systems are investigated. Four protonation steps are evidenced from fluorescence emission studies of hematoporphyrin IX and its more hydrophobic parent compound lacking of alcoholic chain, deuteroporphyrin IX. They are attributed to the successive protonations of the inner nitrogens of the porphyrin cycle (pK = 4.7 and 2.9 for hematoporphyrin and 4.4 and 2.7 for deuteroporphyrin) and successive deprotonations of propionic groups (pK ≈ 5.0 and 5.5 for hematoporphyrin and 5.4 and 6.0 for deuteroporphyrin). These porphyrins, as well as their dimethyl ester forms, are shown to incorporate as monomers into the hydrophobic bilayer of egg phosphatidylcholine small unilamellar vesicles, although the esterified forms are highly aggregated in aqueous solutions. In the case of the non-esterified forms, the incorporation of the porphyrins into the lipidic bilayer is reversible and strongly pH-dependent. A theoretical model is presented which takes into account the various protonation steps and the partition equilibria of the porphyrin between the vesicle lipidic phase and the water medium. The neutral form of the porphyrin (i.e., carboxylic groups protonated) presents the higher affinity, with constants of K ≈ 2 · 10⁵ and K ≈ 6 · 10⁶ M⁻¹ (relative to lipid concentration) for hematoporphyrin and deuteroporphyrin, respectively. Protonation of one inner nitrogen leading to the monocationic form is sufficient to prevent incorporation into the hydrophobic bilayer. On the other hand, deprotonation of the peripheral propionic chains leading to anionic forms is less effective. These interactions between vesicles and porphyrins lead to shifts of the apparent pK of nitrogens and carboxylic groups, the latter one being now in the range of physiological pH. These results are discussed with regards to the hypothesis of a possible role of pH in the preferential uptake of porphyrins by tumors.     

Formation of porphyrin pi cation radical in zinc-substituted horseradish peroxidase

Zinc-substituted horseradish peroxidase is oxidized by K2IrCl6 to a characteristic state which retains one oxidizing equivalent more than the zinc peroxidase. The oxidized enzyme gives an optical absorption spectrum similar to that of compound I of peroxidase and catalase, and a g = 2 electron paramagnetic resonance signal which has an intensity corresponding to the porphyrin content. It is reduced back to the zinc peroxidase by a stoichiometric amount of ferrocyanide or by a large excess of K3IrCl6. From the equilibrium data, the value of E0' for the zinc peroxidase couple is estimated to be 0.74 V at pH 6. The oxidized zinc peroxidase is also formed by the addition of H2O2 or upon illumination with white light. The rate constants for the oxidation by K2IrCl6 and H2O2 at pH 8.0 are 8 x 10(5) and 8 x 10(2) M-1 s-1, respectively. No essential spectral change can be observed when K2IrCl6 is added to the metal-free peroxidase (protoporphyrin--apoperoxidase complex) or to zinc-substituted sperm whale myoglobin.     

Protoporphyrin IX-induced structural and functional changes in human red blood cells,haemoglobin and myoglobin

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin: protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.     

The interaction of tumour-localizing porphyrins with collagen, elastin, gelatin, fibrin and fibrinogen

We have already reported in Balb C mouse transplantable mammary carcinoma, that uroporphyrin I and III are superior as tumour localizers when compared to hematoporphyrin derivative and a derivative thereof, photofrin II. This study compares the binding of porphyrins to proteins which may be found in tumour cells or stroma to investigate whether there is a common binding determinant. Coproporphyrin III and deuteroporphyrin IX which are non-tumour localizing porphyrins, were also part of the comparative study. The interaction of these porphyrins with acid soluble collagen and acid insoluble collagen, elastin, and fibrin was evaluated, and the binding of uroporphyrin isomers I and III and deuteroporphyrin IX to gelatin and fibrinogen, was also determined. The results suggest that collagen, especially the acid soluble form, and gelatin preferentially bind the four porphyrins which localize in mammary carcinoma tissue. The well reported observations that malignant epithelial cells, including breast cancer, produce collagen and contain a rate-limiting enzyme in collagen biosynthesis would support the notion that de novo synthesis of this protein may in part govern the tumour uptake and retention of porphyrins. Elastin, fibrinogen and fibrin showed non-discriminant binding to the porphyrins under study.     

The physical chemistry of Hemes II. Electron paramagnetic resonance study of the interaction of some iron(III)-porphyrins with fluoride ion in dimethylformamide

The interaction of F⁻ with high and low spin ferric deuteroporphyrin IX dimethyl ester and a low spin model compound, bis(histidine methyl ester) deuterohemin IX has been studied in dimethylformamide solution by low-temperature EPR. The reaction of F⁻ with these complexes leads to high spin compounds. The structure of the EPR line at g = 2 is due to superhyperfine interactions with axial fluoride ligands. It allows their identification as mono- or difluoride complexes. Their optical absorption spectra are reported. In the particular cases of bis(imidazole) deuterohemin IX dimethyl ester and of the model compound, the variations of the EPR spectra as functions of concentration of ionic ligand are reported. Three new low spin complexes are thus obtained. They are characterized by a specific interaction of F⁻ with the NH group of the imidazole ring. This is proved following a second independent study in which we report the changes in g tensor principal values of low spin ferric porphyrins with the basicity (pK_a) of various nitrogenous bases.     

Protoporphyrin IX activates the Mg dependent guanylate cyclase from rat liver plasma membranes

In the presence of Mg-GTP, the rat liver guanylate cyclase, in either intact membranes or trypsin solubilized form, was stimulated by protoporphyrin IX 6 to 10-fold. However, when Mn-GTP was the substrate, protoporphyrin IX activated the membrane-bound guanylate cyclase only 50%, in contrast to the marked activation reported for the cytosolic enzyme. Meso- and deuteroporphyrin IX, hematoporphyrin and coproporphyrin III also activated membrane guanylate cyclase while uroporphyrin III, and hemin had no effect. Basal, Mg2+-dependent activity exhibited two classes of catalytic sites with apparent Km values of 2 mM and 0.12 mM. Activation by protoporphyrin resulted in the disappearance of the low affinity sites. The activated enzyme exhibited Michaelis-Menten kinetics and no alteration in its requirement for excess Mg2+. These data indicate that, in the presence of Mg2+, a heme-like structure can interact with the membrane-bound guanylate cyclase and regulate its activity.