Reactivity of the heme-dioxygen complex of the inducible nitric oxide synthase in the presence of alternative substrates.

Single turnover reactions of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) in the presence of several non alpha-amino acid N-hydroxyguanidines and guanidines were studied by stopped-flow visible spectroscopy, and compared with reactions using the native substrates L-arginine (L-arg) or N(omega)-hydroxy-L-arginine (NOHA). In experiments containing dihydrobiopterin, a catalytically incompetent pterin, and each of the studied substrates, L-arg, butylguanidine (BuGua), para-fluorophenylguanidine (FPhGua), NOHA, N-butyl- and N-(para-fluorophenyl)-N'-hydroxyguanidines (BuNOHG and FPhNOHG), the formation of a iron(II) heme-dioxygen intermediate (Fe(II)O2) was always observed. The Fe(II)O2 species then decayed to iron(III) iNOSoxy at rates that were dependent on the nature of the substrate. Identical reactions containing the catalytically competent cofactor tetrahydrobiopterin (BH4), iNOSoxy and the three N-hydroxyguanidines, all exhibited an initial formation of an Fe(II)O2 species that was successively converted to an Fe(III)NO complex and eventually to high-spin iron(III) iNOSoxy. The formation and decay kinetics of the Fe(III)NO complex did not vary greatly as a function of the N-hydroxyguanidine structure, but the formation of Fe(III)NO was substoichiometric in the cases of BuNOHG and FPhNOHG. Reactions between BH4-containing iNOSoxy and BuGua exhibited kinetics similar to those of the corresponding reaction with L-arginine, with formation of an Fe(II)O2 intermediate that was directly converted to high-spin iron(III) iNOSoxy. In contrast, no Fe(II)O2 intermediate was observed in the reaction of BH4-containing iNOSoxy and FPhGua. Multi-turnover reaction of iNOS with FPhGua did not lead to formation of NO or to hydroxylation of the substrate, contrary to reactions with BuGua or L-arg. Our results reveal how different structural and chemical properties of NOS substrate analogues can impact on the kinetics and reactivity of the Fe(II)O2 intermediate, and support an important role for substrate pKa during NOS oxygen activation.

Relationship between the structure of guanidines and N-hydroxyguanidines, their binding to inducible nitric oxide synthase (iNOS) and their iNOS-catalysed oxidation to NO.

The binding of several alkyl- and aryl-guanidines and N-hydroxyguanidines to the oxygenase domain of inducible NO-synthase (iNOS(oxy)) was studied by UV/Vis difference spectroscopy. In a very general manner, monosubstituted guanidines exhibited affinities for iNOS(oxy) that were very close to those of the corresponding N-hydroxyguanidines. The highest affinities were observed for the natural substrates, L-arginine and N(omega)-hydroxy-L-arginine (K(d) at the microm level). The deletion of either the CO2H or the NH2 function of their amino acid moiety led to dramatic decreases in the affinity. However, alkylguanidines with a relatively small alkyl chain exhibited interesting affinities, the best being observed for a butyl chain (K(d) =20 microM). Arylguanidines also bound to iNOS(oxy), however, with lower affinities (K(d) > 250 microm). Many N-alkyl- and N-aryl-N'-hydroxyguanidines are oxidized by iNOS with formation of NO, whereas only few alkylguanidines led to significant production of NO under identical conditions, and all the arylguanidines tested to date were unable to lead to the production of NO. The k(cat) values of NO production from the oxidation by iNOS of the studied N-hydroxyguanidines were found to vary independently of their affinity for the protein. The k(cat) values determined for the two-step oxidation of alkylguanidines to NO were not clearly related to the K(d) of these substrates toward iNOS(oxy). However, there is a qualitative relationship between these k(cat) values and the apparent rate constants of dissociation of the complex between iNOS(oxy) and the corresponding N-alkyl-N'-hydroxyguanidine (k(off) (app)) that were determined by stopped-flow UV/Vis spectroscopy. These data indicate that a key factor for efficient oxidation of a guanidine by iNOS to NO is the ability of the corresponding N-hydroxyguanidine to bind to the active site without being too rapidly released before its further oxidation. This explains why 4,4,4-trifluorobutylguanidine is so far the best non-alpha-amino acid guanidine substrate of iNOS with formation of NO, because the k(off) (app) of the corresponding N-hydroxyguanidine is particularly low. This suggests that the rational design of guanidines as new NO donors upon in situ oxidation by NOSs should take into account both thermodynamic and kinetic characteristics of the interaction of the protein not only with the guanidine but also with the corresponding N-hydroxyguanidine.

N-Aryl N'-hydroxyguanidines, a new class of NO-donors after selective oxidation by nitric oxide synthases: structure-activity relationship.

The formation of nitric oxide (NO) was followed during the oxidation of 37 N-hydroxyguanidines or related derivatives, including 18 new N-aryl N'-hydroxyguanidines, by recombinant inducible nitric oxide synthase (NOS II). Several N-aryl N'-hydroxyguanidines bearing a relatively small, electron-donating para subtituent, such as H, F, Cl, CH(3), OH, OCH(3), and NH(2), led to NO formation rates between 8 and 41% of that of NO formation from the natural NOS substrate, N(omega)-hydroxy-L-arginine (NOHA). The characteristics of these reactions were very similar to those previously reported for the oxidation of NOHA by NOS:(i) the strict requirement of NOS containing (6R)-5,6,7,8-tetrahydro-L-biopterin, reduced nicotinamide adenine dinucleotide phosphate, and O(2) for the oxidation to occur, (ii) the formation of NO and the corresponding urea in a 1:1 molar ratio, and (iii) a strong inhibitory effect of the classical NOS inhibitors such as N(omega)-nitro-L-arginine and S-ethyl-iso-thiourea. Structure-activity relationship studies showed that two structural factors are crucial for NO formation from compounds containing a C(triple bond)NOH function. The first one is the presence of a monosubstituted N-hydroxyguanidine function, since disubstituted N-hydroxyguanidines, amidoximes, ketoximes, and aldoximes failed to produce NO. The second one is the presence of a N-phenyl ring bearing a relatively small, not electron-withdrawing para substituent that could favorably interact with a hydrophobic cavity close to the NOS catalytic site. The k(cat) value for NOS II-catalyzed oxidation of N-para-fluorophenyl N'-hydroxyguanidine was 80% of that found for NOHA, and its k(cat)/K(m) value was only 9-fold lower than that of NOHA. Interestingly, the K(m) value found for NOS II-catalyzed oxidation of N-(3-thienyl) N'-hydroxyguanidine was 25 microM, almost identical to that of NOHA. Recombinant NOS I and NOS III also oxidize several N-aryl N'-hydroxyguanidines with the formation of NO, with a clearly different substrate specificity. The best substrates of the studied series for NOS I and NOS III were N-(para-hydroxyphenyl) and N-(meta-aminophenyl) N'-hydroxyguanidine, respectively. Among the studied compounds, the para-chlorophenyl and para-methylphenyl derivatives were selective substrates of NOS II. These results open the way toward a new class of selective NO donors after in situ oxidation by each NOS family.

First non-alpha-amino acid guanidines acting as efficient NO precursors upon oxidation by NO-synthase II or activated mouse macrophages.

A study of the oxidation of a series of guanidines related to L-arginine (L-Arg) and of various alkyl- and arylguanidines, by recombinant NO-synthase II (NOS II), led us to the discovery of the first non-alpha-amino acid guanidine substrate of NOS, acting as an efficient NO precursor. This compound, 3-(trifluoromethyl)propylguanidine, 4, led to a rate of NO formation (k(cat) = 220 +/- 50 min(-1)) only 2 times lower than that of L-Arg. Formation of 1 mol of NO upon NOS II-catalyzed oxidation of 4 occurred with consumption of 2.9 mol of NADPH, which corresponds to a 52% coupling between electron transfer and oxygenation of its guanidine function. Its oxidation by activated mouse macrophages in an L-Arg-free medium resulted in NO(2)(-) formation that was inhibited by classical NOS inhibitors with a rate only 2-3 times lower than that observed with L-Arg itself. These results open the way toward the research of selective, stable guanidine substrates of NOS that could be interesting, new NO donors after in situ oxidation by a given NOS isoform.

Two modes of binding of N-hydroxyguanidines to NO synthases: first evidence for the formation of iron-N-hydroxyguanidine complexes and key role of tetrahydrobiopterin in determining the binding mode.

The interaction of various N-alkyl- and N-aryl-N'-hydroxyguanidines with recombinant NOS containing or not containing tetrahydrobiopterin (BH(4)) was studied by visible, electronic paramagnetic resonance (EPR), and resonance Raman (RR) spectroscopy. N-Hydroxyguanidines interact with the oxygenase domain of BH(4)-free inducible NOS (BH(4)-free iNOS(oxy)), depending on the nature of their substituent, with formation of two types of complexes that are characterized by peaks around 395 (type I) and 438 nm (type II') during difference visible spectroscopy. The complex formed between BH(4)-free iNOS(oxy) and N-benzyl-N'-hydroxyguanidine 1 (type II') exhibited a Soret peak at 430 nm, EPR signals at g = 1.93, 2.24, and 2.38, and RR bands at 1374 and 1502 cm(-)(1) that are characteristic of a low-spin hexacoordinated Fe(III) complex. Analysis of its EPR spectrum according to Taylor's equations indicates that the cysteinate ligand of native BH(4)-free iNOS(oxy) is retained in that complex. Similar iron(III)-ligand complexes were formed upon reaction of 1 and several other N-hydroxyguanidines with BH(4)-free full-length iNOS and BH(4)-free nNOS(oxy). However, none of the tested N-hydroxyguanidines were able to form such iron(III)-ligand complexes with BH(4)-containing iNOS(oxy), indicating that a major factor involved in the mode of binding of N-hydroxyguanidines to NOS is the presence (or absence) of BH(4) in their active site. Another factor that plays a key role in the mode of binding of N-hydroxyguanidines to NOS is the nature of their substituent. The N-hydroxyguanidines bearing an N-alkyl substituent exclusively or mainly led to type II' iron-ligand complexes. Those bearing an N-aryl substituent mainly led to type II' complexes, even though some of them exclusively led to type I complexes. Interestingly, the K(s) values calculated for BH(4)-free iNOS(oxy)-N-hydroxyguanidine complexes were always lower when their substituents bore an aryl group (140-420 microM instead of 1000-3900 microM), suggesting the existence of pi-pi interactions between this group and an aromatic residue of the protein. Comparison of the spectral and physicochemical properties of the N-hydroxyguanidine complexes of BH(4)-free iNOS(oxy) (type II') with those of the previously described corresponding complexes of microperoxidase (MP-8) suggests that, in both cases, N-hydroxyguanidines bind to iron(III) via their oxygen atom after deprotonation or weakening of the O-H bond. The aforementioned results are discussed in relation with recent data about the transient formation of iron-product intermediates during the catalytic cycle of l-arginine oxidation by eNOS. They suggest that N-hydroxyguanidines could constitute a new class of good ligands of heme proteins.