| Abstract
| - The interaction of various N-alkyl- and N-aryl-N‘-hydroxyguanidines with recombinant NOScontaining or not containing tetrahydrobiopterin (BH4) was studied by visible, electronic paramagneticresonance (EPR), and resonance Raman (RR) spectroscopy. N-Hydroxyguanidines interact with theoxygenase domain of BH4-free inducible NOS (BH4-free iNOSoxy), depending on the nature of theirsubstituent, with formation of two types of complexes that are characterized by peaks around 395 (typeI) and 438 nm (type II‘) during difference visible spectroscopy. The complex formed between BH4-freeiNOSoxy and N-benzyl-N‘-hydroxyguanidine 1 (type II‘) exhibited a Soret peak at 430 nm, EPR signals atg = 1.93, 2.24, and 2.38, and RR bands at 1374 and 1502 cm-1 that are characteristic of a low-spinhexacoordinated Fe(III) complex. Analysis of its EPR spectrum according to Taylor's equations indicatesthat the cysteinate ligand of native BH4-free iNOSoxy is retained in that complex. Similar iron(III)−ligandcomplexes were formed upon reaction of 1 and several other N-hydroxyguanidines with BH4-free full-length iNOS and BH4-free nNOSoxy. However, none of the tested N-hydroxyguanidines were able to formsuch iron(III)−ligand complexes with BH4-containing iNOSoxy, indicating that a major factor involved inthe mode of binding of N-hydroxyguanidines to NOS is the presence (or absence) of BH4 in their activesite. Another factor that plays a key role in the mode of binding of N-hydroxyguanidines to NOS is thenature of their substituent. The N-hydroxyguanidines bearing an N-alkyl substituent exclusively or mainlyled 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 Ks values calculated forBH4-free iNOSoxy−N-hydroxyguanidine complexes were always lower when their substituents bore anaryl group (140−420 μM instead of 1000−3900 μM), suggesting the existence of π−π interactions betweenthis group and an aromatic residue of the protein. Comparison of the spectral and physicochemical propertiesof the N-hydroxyguanidine complexes of BH4-free iNOSoxy (type II‘) with those of the previously describedcorresponding complexes of microperoxidase (MP-8) suggests that, in both cases, N-hydroxyguanidinesbind to iron(III) via their oxygen atom after deprotonation or weakening of the O−H bond. Theaforementioned 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 thatN-hydroxyguanidines could constitute a new class of good ligands of heme proteins.
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