Loss of atomic nitrogen from even-electron ions? A study on benzodiazepines.

The fragment spectra of protonated nitro-substituted benzodiazepines show an unusual fragment [M + H - 14]+ , which is shown by accurate mass measurement to be due to the loss of a nitrogen atom. Our investigations show that this apparent loss of atomic nitrogen is rather an attachment of molecular oxygen to the [M + H - NO2 ]+• ion, which is the main fragment ion in these spectra. The oxygen attachment is exothermic, and rate constants have been derived. MSn spectra show that it is not easily reversible upon fragmentation of the adduct ion and that it is also observed with some secondary and tertiary fragments, which allows to limit the attachment site to the aromatic ring annulated to the diazepine moiety. Fragments of the oxygen adduct ion indicate that the O2 molecule dissociates in the adduct formation process, and the two oxygen atoms are bound to different sites of the ion. Comparison with radical cations generated by fragmentation of non-nitro-substituted benzodiazepines, none of which showed an oxygen attachment, and the fragmentation mechanisms involved in their formation indicates that the [M + H - NO2 ]+• ion is a distonic ion with the charge and radical site neighbored on the aromatic ring. From these results, we derive a proposal for the formation and structure of the [M + H - NO2 + O2 ]+• ion, which explains the experimental observations. Copyright © 2015 John Wiley & Sons, Ltd.

The oxygen-independent coproporphyrinogen III oxidase HemN utilizes harderoporphyrinogen as a reaction intermediate during conversion of coproporphyrinogen III to protoporphyrinogen IX.

During heme biosynthesis the oxygen-independent coproporphyrinogen III oxidase HemN catalyzes the oxidative decarboxylation of the two propionate side chains on rings A and B of coproporphyrinogen III to the corresponding vinyl groups to yield protoporphyrinogen IX. Here, the sequence of the two decarboxylation steps during HemN catalysis was investigated. A reaction intermediate of HemN activity was isolated by HPLC analysis and identified as monovinyltripropionic acid porphyrin by mass spectrometry. This monovinylic reaction intermediate exhibited identical chromatographic behavior during HPLC analysis as harderoporphyrin (3-vinyl-8,13,17-tripropionic acid-2,7,12,18-tetramethylporphyrin). Furthermore, HemN was able to utilize chemically synthesized harderoporphyrinogen as substrate and converted it to protoporphyrinogen IX. These results suggest that during HemN catalysis the propionate side chain of ring A of coproporphyrinogen III is decarboxylated prior to that of ring B.

The Pseudomonas aeruginosa nirE gene encodes the S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase required for heme d(1) biosynthesis.

Biosynthesis of heme d(1), the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd(1), requires the methylation of the tetrapyrrole precursor uroporphyrinogen III at positions C-2 and C-7. We produced Pseudomonas aeruginosa NirE, a putative S-adenosyl-L-methionine (SAM)-dependent uroporphyrinogen III methyltransferase, as a recombinant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography. Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer. NirE was shown to be a SAM-dependent uroporphyrinogen III methyltransferase that catalyzes the conversion of uroporphyrinogen III into precorrin-2 in vivo and in vitro. A specific activity of 316.8 nmol of precorrin-2 h(-1) x mg(-1) of NirE was found for the conversion of uroporphyrinogen III to precorrin-2. At high enzyme concentrations NirE catalyzed an overmethylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin. Substrate inhibition was observed at uroporphyrinogen III concentrations above 17 microM. The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent uroporphyrinogen III methyltransferases involved in siroheme and cobalamin biosynthesis. A P. aeruginosa nirE transposon mutant was not complemented by native cobA encoding the SAM-dependent uroporphyrinogen III methyltransferase involved in cobalamin formation. However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special requirement of NirE for heme d(1) biosynthesis.

Human cardiac phospholipase D activity is tightly controlled by phosphatidylinositol 4,5-bisphosphate.

Phospholipase D (PLD) plays a central role in receptor-mediated breakdown of choline phospholipids and formation of phosphatidic acid (PA), an important regulator of cardiac function. However, specific mechanisms that regulate myocardial PLD activity remain largely unknown, particularly in the human heart. We hypothesized that phosphatidylinositol 4,5-bisphosphate (PIP2), best known as substrate for phospholipase C (PLC) isozymes, plays a critical role in regulating myocardial PLD activity. We examined the effect of PIP2 on human myocardial PLD activity in vitro by utilizing a fluorescence HPLC assay. PIP2 increased 10-fold the maximal activity of a partially solubilized PLD from human atrial myocardium. PIP2-stimulated PLD activity was accompanied by a consecutive increase in diacylglycerol, indicating dephosphorylation of PA by PA phosphohydrolase. Likewise, phosphatidylinositol 3,4,5-trisphosphate, which is produced from PIP2 by phosphatidylinositol 3-kinase, increased PLD activity with about the same potency but with somewhat lower efficacy. In contrast, other phospholipids were ineffective, indicating that the action of PIP2 on PLD is highly specific. Neomycin, a high-affinity ligand of PIP2, inhibited PLD activity in human atrial myocardium, but had no effect on the activity of partially solubilized enzyme. The addition of PIP2 restored the sensitivity of solubilized PLD to neomycin inhibition, indicating that neomycin inhibits PLD activity by binding to endogenous PIP2. Our results demonstrate a critical role for PIP2 in human cardiac PLD activity and suggest that PIP2 synthesis (by phosphatidylinositol 4-phosphate 5-kinase) and hydrolysis (by PIP2-specific PLC) could be important determinants in regulating PLD signal transduction in the human heart.

Adrenergic activation of cardiac phospholipase D: role of alpha(1)-adrenoceptor subtypes.

OBJECTIVE: Adrenergic stimulation of the heart leads to activation of the phospholipase D signal transduction pathway with formation of the intracellular second messengers phosphatidic acid and diacylglycerol, which may play a role in the development of myocardial hypertrophy by activating mitogen-activated protein kinases and protein kinase C. So far, the adrenergic receptor subtypes mediating activation of cardiac phospholipase D are not known. METHODS: We developed an assay for determination of phospholipase D activity in the isolated perfused rat heart. Utilizing the phospholipase D specific transphosphatidylation reaction the stable product phosphatidylethanol (PEtOH) is formed in rat hearts perfused in the presence of 1% ethanol. Myocardial PEtOH formation was used as a marker of phospholipase D activity and was determined by HPLC and evaporative light-scattering detection (PEtOH microg/mg myocardial protein). RESULTS: Basal PEtOH formation in unstimulated hearts was 0.06+/-0.01 microg/mg. Stimulation of the hearts with norepinephrine resulted in a concentration-dependent phospholipase D activation with a maximum formation of PEtOH (0.17+/-0.01 microg/mg) at 100 micromol/l norepinephrine. The norepinephrine-induced increase in PLD activity was completely blocked by the alpha(1)-adrenoceptor antagonist prazosin and was unaffected by the beta-adrenoceptor antagonist propranolol. Further characterisation of alpha(1)-adrenoceptor subtypes with selective alpha(1)-adrenoceptor antagonists demonstrated a complete inhibition of the norepinephrine-induced phospholipase D activation by WB 4101 (alpha(1A)-selective: 0.06+/-0.01 microg/mg) and by BMY 7378 (alpha(1D)-selective: 0.07+/-0.01 microg/mg). In contrast, the alpha(1B)-adrenoceptor antagonist chloroethylclonidine had no inhibitory effect on norepinephrine-stimulated phospholipase D activity (0.14+/-0.01 microg/mg). CONCLUSION: Adrenergic activation of the cardiac phospholipase D signal transduction pathway is mediated by alpha(1)-adrenoceptors. Here, the alpha(1A)-adrenoceptor subtype, but not the alpha(1B)-adrenoceptor are coupled to activation of cardiac phospholipase D.