Substrate-specific Interactions with the Heme-bound Oxygen Molecule of Nitric-oxide Synthase

François J.M Chartier,Manon Couture

doi:10.1074/jbc.m701800200

Abstract

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.

Highlights

To carry out these two reactions, the heme iron at the active site of Nitric-oxide synthases (NOSs) must first be reduced to be able to bind molecular oxygen (O2) and to form an oxygenated complex (FeIIO2)
To complement our first study of the FeIIO2 complex of Staphylococcus aureus NOS (saNOS) in the presence of L-arginine [23], we report an investigation of the interactions of the FeIIO2 complex in the presence of NOHA for saNOS and Bacillus subtilis NOS by resonance Raman spectroscopy
This effect of L-arginine correlates with the increased stability of the FeIIO2 complex, which decays at rates of 5.6 and 1 sϪ1 compared with 39.6 sϪ1 without L-arginine [23]

Summary

Introduction

To carry out these two reactions, the heme iron at the active site of NOSs must first be reduced to be able to bind molecular oxygen (O2) and to form an oxygenated complex (FeIIO2). Differences in the frequencies of the ␯Fe–O and ␯O–O modes were observed in the presence of NOHA with respect to those measured with L-arginine, indicating that the hemebound O2 is involved in substrate-specific interactions in both NOSs. The implications of these results for the mechanisms of oxygen activation by NOSs are discussed.

Results

Conclusion