The reaction of nitric oxide synthase (NOS) with oxygen is fast and takes place within several steps, separated by ephemeral inter-mediates. The use of extreme experimental conditions, such as low tell, peratUrC and high pressure, associated to rapid kinetic analysis, has proven to be a convenient tool to study this complex reaction. Stopped-flow experiments under high pressure indicated that oxygen binding occurred in more than one step. This was further corroborated by the detection of two short-lived oxy-compounds, differing in their spectral and electronic properties. Oxy-I resembles the ferrous oxygen complex known for cytochrome P450, whereas oxy-II appears to be locked ill the Superoxide form. Subzero temperature spectroscopy, together with an analytical separation method, revealed that the subsequent one-electron reduction of the oxygen complex is carried Out by the NOS cofactor tetrahydrobiopterin (BH4). The low-temperature stabilized oxidation product of BH4 was found to be it protonated BH3 radical. Finally, work in the presence of a BH4 analog indicated that proton transfer to the activated oxygen complex is a second essential function of BH4. (c) 2005 Elsevier Inc. All rights reserved.