Abstract
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.
Highlights
Despite much effort, the reaction mechanism of these steps is still unclear
This distinction is not clear-cut; in some cases, low wavelength maxima are found by rapid scan spectroscopy [11], and high wavelength maxima are found by low temperature UV-visible spectroscopy [9]
Detection of Two Spectrally Distinct Oxygen Complexes— Depending on experimental conditions, rapid mixing of reduced eNOSoxy with oxygen-containing buffer resulted in two different spectral species
Summary
The reaction mechanism of these steps is still unclear. Even the spectral properties of the oxyferrous complex are not yet defined; conflicting observations report absorbance maxima differing by up to 15 nm. The strongest differences are those between observations at cryogenic temperatures yielding maxima in the 417– 419-nm region [7,8,9] and observations at higher temperatures by rapid scan techniques yielding considerably red-shifted maxima (430 – 432 nm) (10 –14) This distinction is not clear-cut; in some cases, low wavelength maxima are found by rapid scan spectroscopy [11], and high wavelength maxima are found by low temperature UV-visible spectroscopy [9]. The formation of NO is catalyzed by nitric oxide synthases (NOS; EC 1.14.13.39) via a conditions such as temperature and presence of substrate and pteridine cofactor, two different heme-oxy complexes are formed. These complexes are further distinguished by their ability to exchange oxygen by CO at Ϫ30 °C. The dependence of these oxy-complexes on other reaction partners may be important in the mechanism of uncoupling of NADPH oxidation from product formation
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