N-hydroxyguanidines as new heme ligands: UV-visible, EPR, and resonance Raman studies of the interaction of various compounds bearing a C=NOH function with microperoxidase-8.

Abstract

Interaction between microperoxidase-8 (MP8), a water-soluble hemeprotein model, and a wide range of N-aryl and N-alkyl N'-hydroxyguanidines and related compounds has been investigated using UV-visible, EPR, and resonance Raman spectroscopies. All the N-hydroxyguanidines studied bind to the ferric form of MP8 with formation of stable low-spin iron(III) complexes characterized by absorption maxima at 405, 535, and 560 nm. The complex obtained with N-(4-methoxyphenyl) N'-hydroxyguanidine exhibits EPR g-values at 2.55, 2.26, and 1.86. The resonance Raman (RR) spectrum of this complex is also in agreement with an hexacoordinated low-spin iron(III) structure. The dissociation constants (K(s)) of the MP8 complexes with mono- and disubstituted N-hydroxyguanidines vary between 15 and 160 microM at pH 7.4. Amidoximes also form low-spin iron(III) complexes of MP8, although with much larger dissociation constants. Under the same conditions, ketoximes, aldoximes, methoxyguanidines, and guanidines completely fail to form such complexes with MP8. The K(s) values of the MP8-N-hydroxyguanidine complexes decrease as the pH of the solution is increased, and the affinity of the N-hydroxyguanidines toward MP8 increases with the pK(a) of these ligands. Altogether these results show that compounds involving a -C(NHR)=NOH moiety act as good ligands of MP8-Fe(III) with an affinity that depends on the electron-richness of this moiety. The analysis of the EPR spectrum of the MP8-N-hydroxyguanidine complexes according to Taylor's equations shows a strong axial distortion of the iron, typical of those observed for hexacoordinated heme-Fe(III) complexes with at least one pi donor axial ligand (HO(-), RO(-), or RS(-)). These data strongly suggest that N-hydroxyguanidines bind to MP8 iron via their oxygen atom after deprotonation or weakening of their O-H bond. It thus seems that N-hydroxyguanidines could constitute a new class of strong ligands for hemeproteins and iron(III)-porphyrins.