FULL PAPER z Biosynthesis of Nitric Oxide-Quantum Chemical Modelling of N"-Hydroxy-L-arginine Formation Ewa Wasielewska, Malgorzata Witko, Graiyna Stochel and Zofia Stasicka* Abstract: zyxwvutsrqpon The electronic structure (charge distribution, bond indices, character of the frontier orbitals) and geometry (bond distances and angles) of L-arginine and N-methyl-L-arginine were determined by means of the INDO procedure. The method was also adopted to model the conversion of L-arginine into N-hydroxy- L-arginine in biological systems. This re- vcaled that the approach of diatomic 0 species does not result in reaction, where- as the approach of either an 0 atom or an 02- ion leads to insertion of oxygen and formation of hydroxy-L-arginine. The in- sertion of oxygen between the nitrogen and hydrogen atoms lcads to more stable products than insertion into the C-H bond. The same results were obtained for N-methyl-L-arginine, and are consistent Keywords amino acids zyxwvut * biosynthesis hydroxyl- ations * nitric oxides * semiempirical calculations Introduction One of the most exciting findings in biological chemistry during last few years was the discovery that nitric oxide, a potentially toxic molecule, is responsible for an astonishing range of phys- iological processes in humans." -41 For cxample, it plays a role in vascular system regulation, nervous system mediation and immunological activity. The biological sources of nitric oxide are endogenous as well as exogenous. The conversion of L-arginine (L-Arg) to NO and ~-citrulline (Cit), catalyzed by a family of homodimeric dioxy- genases called NO synthetases (NOS, EC 1.14.13.39),[439-'11 is a widely accepted hypothesis for the endogenous synthcsis of nitric oxide." -"I Compounds related to arginine (e.g. zyxwvuts N- methyl-L-arginine, N-nitro-L-arginine, N-cyclopropyl-L-arginine) are effective inhibitors of NO synthetases. Four cofactors (heme, FMN, FAD and H,biopterin), two cosubstrates, an oxidant (0,) and a reducer (NADPH) participate in the biosyn- thesis of NO from L-arginine. Isotopic studies have shown that nitric oxide is derived from one of the two equivalent guanidine nitrogen atoms of ~-arginine['. 61 and that dioxygen is the source [*I Prof. Dr. zyxwvutsrqponmlk Z. Stasicka. Prof. Dr. G. Stochel, Dr. E. Wasielewska Department of Inorganic Chemistry, Jagiellonian University ingnrdena 3. 30-060 Krakbw (Poland) Fax: Int. code +(12)335-392 e-mail: zyxwvutsrqpon stasicka@trurl.ch.uj.edu.pl Prof. Dr. M. Witko Institute of Catalysis and Surface Chemistry Polish Academy of Sciences, Krakbw (Poland) with the hypothesis that the inhibitive effect of N-substitution in L-arginine is of no importance for the first step in the biosynthcsis of NO (hydroxylation pro- cess). The mechanistic considerations based on the charge distribution and fron- tier orbital characteristics led to the con- clusion that the most probable mecha- nism of L-arginine hydroxylation consists in electrophilic attack of [Fe0I3+ at the Nu-H bond, initiated by the reduction of L-arginine', followed by insertion of oxy- gen and product oxidation. of oxygen atoms incorporated into NO and ~-citrulline.[~I It has also been shown that thc first intermediate in the reaction is N-hydroxy-L-arginine (NOH-Arg) .['I Furthcrmore, when NOH-Arg is N-labelled on the NH-OH nitrogen, NO is exclu- sively derived from this nitrogen.r8s 'I1 NOS-catalyzed conver- sion of L-arginine to L-citrulline and NO is thus a 5-electron oxidation of one of the guanidine nitrogen atoms of L-arginine. Production of 1 mole of NO consumes 2 moles of 0, (8-electron oxidation) and 1.5 mole NADPH (3-electron reduction), which constitutes a 5-electron oxidation system [Eq. (l)], -3 +2 3H + zyxwv 20, + >C=NH - H,O + NO + >C=O (1) A generally accepted working hypothesis assumes that the reactive form of arginine in aqueous solution at pH zy 2 7 is a monovalent cation with an ionized amino acid group and that the NO synthesis proceeds in two steps (see Schemc 1) .I2. ' ~ "I The first step, an overall two-electron oxidation, is a hydrox- ylation resulting in the formation of N-hydroxy-L-arginine as an enzyme-bound intermediate. The second step, an overall 3-elec- tron oxidation, involves electron removal, oxygen insertion and carbon-nitrogen bond scission to form L-citrulline and an NO free radical. Mechanistic speculations on the biosynthesis of nitric oxide have been based mainly on information of the pri- mary structure of NOS.['. ', ' ~ Each NOS subunit is com- posed of reductase and oxygenase I7l The reduc- tase domain contains binding sites for NADPH, FAD and FMN. The oxygenase domain is presumed to contain binding Chrm. Eur. J 1997, 3, No. zyxwvutsrqponml 4 zyxwvutsrqpon 0 VCH ~~rlu~.~~cscll.srhuf~ mhH, 0.69451 Webiheinz, iY97 (1947-(j539:97!0304-06/)9 $ zyxwv 17.50+ S0/0 609