Yann Henry

Cytochrome P450 catalyzes the oxidation of Nω-hydroxy-L-arginine by NADPH and O2 to nitric oxide and citrulline

Rat liver microsomes catalyze the oxidative denitration of N omega-hydroxy-L-arginine (NOHA) by NADPH and O2 with formation of citrulline and nitrogen oxides like NO and NO2-. Besides NO2- and citrulline, whose simultaneous formation is linear for at least 20 min, the formation of NO could be detected under the form of its P450 and P420-Fe(II) complexes by UV-visible and EPR spectroscopy. Classical inhibitors of NO-synthases, like N omega-methyl-and N omega-nitro-arginine, fail to inhibit the microsomal oxidation of NOHA to citrulline and NO2-. On the contrary classical inhibitors of hepatic cytochromes P450 like CO, miconazole, dihydroergotamine and troleandomycin, strongly inhibit this monooxygenase reaction. These results show that the oxygenation of NOHA by NADPH and O2 with formation of citrulline and NO can be efficiently catalyzed by cytochromes P450 (with rates up to 1.5 turnovers per min for the cytochromes of the 3A subfamily).

Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase

Present knowledge of the different enzymatic steps of the denitrification chains in various bacteria, particularly Paracoccus denitrificans and Pseudomonas aeruginosa has been briefly reviewed. The question whether nitric oxide (NO), nitrous oxide (N2O) and other nitrogen derivatives are obligatory intermediates has been discussed. The second part is an extensive review of the structure and the function of a key enzyme in denitrification, cytochrome c551-nitrite-oxidoreductase from P. aeruginosa. Recent results on the stoichiometry of nitrite reduction have been discussed.

Formation of nitric oxide by cytochrome P450-catalyzed oxidation of aromatic amidoximes

Rat liver microsomes catalyze the oxidation of para-hexyloxy-benzamidoxime 1 to the corresponding arylamide 2 and NO2-, by NADPH and O2. Involvement of cytochromes P450 as catalysts of this reaction was shown by the strong inhibitory effects of CO and miconazole and the spectacular increase of the activity upon treatment of rats with dexamethasone, a specific inducer of cytochromes P450 of the 3A subfamily. Formation of NO during oxidation of 1 was shown by detection of the formation of cytochrome P450- and cytochrome P420-Fe(II)-NO complexes by visible and EPR spectroscopy. The formation of these complexes should be responsible, at least in part, for the fast decrease of the rate of microsomal oxidation of 1 with time. These results suggest that exogenous compounds containing amidine or amidoxime functions could act as precursors of NO in vivo after in situ oxidation by cytochromes P450.

Basic Chemistry of Nitric Oxide and Related Nitrogen Oxides

The general purpose of this chapter is to briefly recall the basic chemistry of NO•, especially its own paramagnetism and that of some of the complexes it forms with transition metals. As a full review is out of our scope, we shall only mention data that could be useful to understand the following chapters. In particular we shall attempt to compile some kinetic data which can enable us to propose whether a given reaction between NO• and a molecular target could be relevant to the biology of a cell or an animal.

Stoichiometry of nitrite reduction catalyzed by Pseudomonas aeruginosa nitrite-reductase

The stoichiometry of the reduction of nitrite catalyzed by Pseudomonas aeruginosa nitrite-reductase (cytochrome cd1) has been shown to yield nitrous oxide as the final product. Gas chromatography experiments demonstrated that nitric oxide is also formed as a free intermediate. A sequential formation of NO and N2O is discussed as opposed to the parallel formation of the two products.

Radical scavenging and electron-transfer reactions in Polyporus Versicolor laccase: A pulse radiolysis study

The interaction of the radicals OH, t-BuO, Eaq, CO2 and O2 with the copper oxidase, laccase, from Polyporus, has been studied by the pulse-radiolysis technique. Each of these radicals formed transient adducts with a broad absorption maximum around 310 nm. Analysis of the optical properties and of the very fast rates of formation of these compounds shows that each radical interacts with a limited number of sites on the polypeptide part of the protein amongst R-S-S-R, histidine and aromatic residues. Interaction with the carbonyl group of some of the peptides bonds is also possible. The few target sites are probably hit simultaneously and electron transfer between these sites may also occur. In all cases, ina subsequent step, intramolecular electron transfer from the polypeptide radical adducts leads to a partial reduction of the blue type-1 Cu2+ with rates varying between 10(3) adn 10(4) s-1. Further reduction of the type-1 CU2+ occurs through a slow intermolecular reaction between two laccase radical transient adducts. In the case of CO2 and O2, this slow reduction could alternatively be due to an intermolecular reaction between laccase and CO2 or O2. The oxidation radicals OH, Br2 and (SCN)2, which formed radical adducts with fully ascorbate-reduced laccase, did not induce any type-1 copper reoxidation.

Enzymatic Targets of Nitric Oxide as Detected by EPR Spectroscopy within Mammal Cells

We concluded in chapter 6 that all metalloproteins are potential targets of NO, as proven by EPR spectroscopy, which is too unspecific to be a really interesting statement. We now turn to some more biologically relevant aspects of NO binding. Its production from L-arginine catalyzed by the inducible NO-synthase (iNOS, NOS II) in murine macrophages and their tumoral target cells was the simplest case to characterize at the molecular level by EPR spectroscopy. [FeS]-containing proteins and ribonucleotide reductase responsible for basic vital cellular functions such as mitochondrial respiration and DNA replication were the earliest enzymatic targets characterized in whole cultured mammal cells.

EPR Detection of Nitrosylated Compounds : Introduction with some Historical Background

Considered for a long time as a simple chemical of great industrial importance, then in turn as a poison and a pollutant, more recently as an important endogenous and ubiquitous gas in mammals, possibly as a “miraculous” gas to use in neonatal intensive care units, etc., nitric oxide has become as fascinating a molecule as molecular oxygen. The main difference between the two gases lies in the facts that we breathe air from our first cry at birth and have some culture-based understanding of air breathing, while the understanding of the various biological roles of NO, such as in the opening of the bronchial alveoles that precedes the first cry of the neonate, is still in its infancy. We think it interesting to hint at the various sources of NO on earth.