Evidence of two distinct oxygen complexes of reduced endothelial nitric oxide synthase

Oxygen binding to the oxygenase domain of reduced endothelial nitric oxide synthase (eNOS) results in two distinct species differing in their Soret and visible absorbance maxima and in their capacity to exchange oxygen by CO. At 7 degrees C, heme-oxy I (with maxima at 420 and 560 nm) is formed very rapidly (k(on) approximately 2.5.10(6) m(-1).s(-1)) in the absence of substrate but in the presence of pterin cofactor. It is capable of exchanging oxygen with CO at -30 degrees C. Heme-oxy II is formed more slowly (k(on) approximately equal to 3.10(5) m(-1).s(-1)) in the presence of substrate, regardless of the presence of pterin. It is also formed in the absence of both substrate and pterin. In contrast to heme-oxy I, it cannot exchange oxygen with CO at cryogenic temperature. In the presence of arginine, heme-oxy II is characterized by absorbance maxima near 432, 564, and 597 nm. When arginine is replaced by N-hydroxyarginine, and also in the absence of both substrate and pterin, its absorbance maxima are blue-shifted to 428, 560, and 593 nm. Heme-oxy I seems to resemble the ferrous dioxygen complex observed in many hemoproteins, including cytochrome P450. Heme-oxy II, which is the oxygen complex competent for product formation, appears to represent a distinct conformation in which the electronic configuration is essentially locked in the ferric superoxide complex.