A Weak Fe–O Bond in the Oxygenated Complex of the Nitric-oxide Synthase of Staphylococcus aureus

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 H4B. 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 (FeIIIO2-) with a single νO–O mode at 1135 cm–1. In the presence of l-arginine, an additional νO–O mode at 1123 cm–1 was observed, indicating an increased π back-bonding electron donation to the bound oxygen induced by the substrate. With saNOS, this is the first time that the νFe–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 P450cam. 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.