L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen.

Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water.


Previously
proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO') synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO' synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H~180/1602. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry.
Its mass spectrum matched that of L-citrulline generated in Hz160/1602. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO' synthase derives from dioxygen and not from water. Synthesis of nitric oxide (NO')' is a recently discovered property of macrophages (2)(3)(4), endothelial cells (5, 6), neutrophils (7, a), some tumor cells (9), hepatocytes (lo), and unidentified cells in adrenal gland (11) and cerebellum (12). NO' is responsible for some of the cytotoxic effects of macrophages on tumor cells and microbes (13)(14)(15) and is a principal endothelium-derived relaxing factor (5,6 of NO' in the other cell types are not yet clear. In macrophages and endothelial cells, 15N studies have shown that an NW-guanidino nitrogen of L-arginine is utilized to produce NO', leaving L-citrulline (16)(17)(18). NADPH is absolutely required (19). Recently we (20) and others (21) demonstrated that macrophage NO' synthase requires tetrahydrobiopterin as a cofactor. A reductase to reduce biopterins (e.g. dihydropteridine reductase) is necessary for continuous recycling of the cofactor (20). FAD and a reduced thiol such as GSH are necessary for maximal NO' generation by the partially purified macrophage enzyme (19). The requirement for FAD and NADPH suggests involvement of a flavoprotein (19,22).
Two mechanisms of L-citrulline and NO' formation from L-arginine have been proposed. One involves an enzymecatalyzed hydrolytic deimination of L-arginine to form Lcitrulline and ammonia, with subsequent oxidation of the ammonia to form NO' (3). A second mechanism involves formation of NW-hydroxyl-L-arginine via an NADPH-dependent monooxygenase reaction followed by electron removal and homolytic bond cleavage to yield NO' and an amino acid carbodiimide, which upon hydration forms citrulline (18,21). Both mechanisms predict incorporation of water oxygen to form citrulline. To test this, we studied L-citrulline production by macrophage NO ' synthase in HZ"0 or under an 1802 atmosphere. The results indicate that the oxygen atom in the ureido group of L-citrulline originates from dioxygen, not water. Three major peaks were eluted from the GC (Fig. 1A). Two peaks eluting at -4.5 and -14.4 min showed a similar mass spectrum.* The inset of Fig. 1B shows the mass spectrum of the 14.4-min peak, which was similar to a previously published spectrum of L-citrulline dimethylaminomethylene methyl ester (DMAM-citrulline) (26,27). The spectrum showed the molecular ion (M+) of m/z 299 and a base ion of m/z 99. The base fragment ion was reported to contain the ureido carbonyl group of L-citrulline (27). The 4.5-min peak showed the same base ion of m/z 99 and a similar ion fragmentation pattern (Fig. 1B). However, fragment ions heavier than the ion group at m/z 227 ((M -72)+) were not detected. This substance eluting at -4.5 min may result from the loss of (CH&NCHNH from DMAMcitrulline during derivatization (26). L-Citrulline produced by NO' synthase was partially purified with anion exchange chromatography, derivatized with Methyl-& and analyzed by GC/MS. The 14.4-min peak of DMAM-citrulline was not separated from contaminants. However, the derivatized reaction component eluting at -4.5 min was well separated, and its mass spectrum displayed a base ion of m/z 99 and an ion fragmentation pattern similar to that of authentic citrulline (compare Fig. 1B and Fig. 20). When the enzyme reaction was performed in the presence of diphenyleneiodonium, a recently discovered NO' synthase inhibitor (22), disappearance of the 4.5-min peak correlated with inhibition of NO' synthesis (data not shown). In a similar experiment using N,O-bis(trimethylsilyl)trifluoroacetamide as a derivatizing reagent, an enzyme reaction product showed a retention time and mass spectrum identical to those of authentic L-citrulline.3 Thus, our GC/MS studies confirm that citrulline is a product of NO' synthase, as shown previously by other methods (3,16).

EXPERIMENTAL PROCEDURES
Next, the enzyme reaction was performed in 94% HZ"0 under aerobic conditions (i.e. in an 1602 atmosphere). Citrulline in the reaction product was partially purified, derivatized with Methyl-& and analyzed with GC/MS as before. In the single ion monitoring at m/z 99 and 101, DMAM-citrulline showed a peak of m/z 99 at -4.5 min, while no peak of m/z 101 eluted between 4 and 5 min (Fig. ZB). This peak pattern in single ion monitoring was the same as in the sample from the NO' synthase reaction in H2160/1602 ( Fig. 2A). Also, the ion fragmentation patterns were identical for DMAM-citrulline produced under these two different reaction conditions (Fig. 2, compare D and E). These results suggested that water oxygen was not incorporated into citrulline during the enzyme reaction.
When the NO' synthase reaction was performed anaerobically, production of citrulline was reduced by >98% (rz = 2 experiments), indicating the requirement of oxygen for enzymatic L-citrulline synthesis. Therefore, the enzyme reaction was next performed in the presence of "OZ. Single ion monitoring at m/z 101 detected a peak eluting from the gas chromatograph at -4.5 min (Fig. 2C). The 4.5.min peak of m/z 101 was not detected in parallel experiments run in an i602 atmosphere (Fig. 2, A and B). The mass spectrum of the 4. was performed in "02, the base ion of m/z 100 shifted to 102 (Fig. 3, inset). These results confirmed that dioxygen is incorporated into the citrulline product of NO ' synthase.
Finally, to exclude the possibility of the incorporation of dioxygen into citrulline from an exchange or a side reaction during incubation in an i8O2 environment, NO' synthase was incubated with isOZ and the various cofactors, but L-citrulline (1 mM) replaced L-arginine. t-Citrulline recovered from this incubate displayed the same mass spectrum as that of authentic citrulline not exposed to the enzyme preparation (data not shown). DISCUSSION These observations make two points. First, they establish that dioxygen is a cosubstrate of macrophage NO' synthase. This brings to six the number of cosubstrates and cofactors known to be required for full activity of the partially purified enzyme, namely L-arginine, 02, NADPH, tetrahydrobiopterin, FAD, and thiol. Second, these findings demonstrate that macrophage NO' synthase incorporates dioxygen to form Lcitrulline during NO' synthesis from L-arginine, excluding reaction mechanisms that hinge on water to provide the ureido oxygen.
NO' synthesis from a guanidino nitrogen of L-arginine represents a 5-electron oxidation that presumably occurs as a multistep process. In macrophages, NO' generation requires tetrahydrobiopterin (20,21), a known cofactor for phenylalanine, tyrosine, and tryptophan mixed function hydroxylases (28)(29)(30)). An NADPH-dependent flavoprotein is also involved in macrophage NO' generation (19,22). Thus, oxygen acti-vation by NO' synthase could be accomplished through tetrahydrobiopterin-and/or flavoprotein-dependent steps (31-33). Our results imply that NO' synthase catalyzes a reaction between activated oxygen and the guanidino carbon of Larginine or of an arginine-derived intermediate. Identifying the source of the NO' oxygen4 will help determine if O2 is also utilized to hydroxyiate an N"-guanidino nitrogen, as proposed previously (18,21). ' We tried to trap NO ' or its oxidation products with morpholine as described (16). The generation of nitrosomorpholine (M+ = m/z 116) from authentic NO ' was detectable by GC/MS. In the presence of H21*0/'602, "0 enrichment was only 19%, suggesting that side reactions with water would not preclude using this method to identify the source of oxygen in NO'. However, the trapping was not successful in the reaction with partially purified NO' synthase preparations, even in the presence of superoxide dismutase and catalase to prolong the half-life of NO ' and even though morpholine was not inhibitory to the enzyme reaction (data not shown).