Abstract
Little is known about the intermediates formed during catalysis by nitric-oxide synthase (NOS). We report here the characterization by resonance Raman spectroscopy of the oxygenated complex of the NOS from Staphylococcus aureus (saNOS) as well as the kinetics of formation and decay of the complex. An oxygenated complex transiently formed after mixing reduced saNOS with oxygen and decayed to the ferric enzyme with kinetics that were dependent on the substrate L-arginine and the cofactor H(4)B. The oxygenated complex displayed a Soret absorption band centered at 430 nm. Resonance Raman spectroscopy revealed that it can be described as a ferric superoxide form (Fe(III)O(2)(-)) with a single nu(O-O) mode at 1135 cm(-1). In the presence of L-arginine, an additional nu(O-O) mode at 1123 cm(-1) was observed, indicating an increased pi back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the nu(Fe-O) mode of a NOS has been observed. The low frequency of this mode, at 517 cm(-1), points to an oxygenated complex that differs from that of P450(cam). The electronic structure of the oxygenated complex and the effect of L-arginine are discussed in relation to the kinetic properties of saNOS and other NOS.
Highlights
The two-step reaction involves a first hydroxylation step that converts L-arginine to the N-hydroxy-L-arginine (NOHA) intermediate and a second hydroxylation step that converts NOHA to citrulline and nitric oxide (NO) [3, 5]
We report here the characterization of the oxygenated complex of saNOS by resonance Raman spectroscopy, which provides important information about the electronic properties of this complex formed during catalysis by nitric-oxide synthase (NOS)
Kinetics of Formation and Decay of the Oxygenated Complex—The kinetics of formation and decay of the oxygenated complex of saNOS were determined by stopped-flow optical spectroscopy
Summary
The two-step reaction involves a first hydroxylation step that converts L-arginine to the N-hydroxy-L-arginine (NOHA) intermediate and a second hydroxylation step that converts NOHA to citrulline and NO [3, 5]. Recent studies have shown that saNOS, unlike mammalian NOS, does not need pterin to stabilize the iron-thiolate bond [11] Pterins such as H4B and tetrahydrofolic acid interact with saNOS as shown by the conservation of the heme vibrational deformation modes in the ferric NO complexes of saNOS, nNOS and iNOS [14, 15]. Recent results have revealed that drNOS can catalyze the regiospecific nitration of tryptophan [16] This activity is completely inhibited by H4B, suggesting that it occurs at the cofactor site. The Oxygenated Complex of saNOS genated complexes with a Soret absorption band at 427 nm similar to the heme-oxyII complex of mammalian NOS [9, 10]. We report here the characterization of the oxygenated complex of saNOS by resonance Raman spectroscopy, which provides important information about the electronic properties of this complex formed during catalysis by NOS
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