Binding of XOR to cellular glycosaminoglycans (GAGs) facilitates nitrite reductase activity under oxygenated conditions

Abstract

Under anoxic conditions, XOR catalyzes the one e − reduction of nitrite ( NO 2 - ) to NO at the Mo-cofactor. However, the conditions that favor e − transfer to NO 2 - at the Mo-cofactor over e − transfer to O 2 at the FAD site have not been defined. Using various detection methods, NO 2 - reduction by purified XOR was analyzed in an O 2 controlled environment. At 0% O 2 , XOR reduced NO 2 - , generating 17.0 ± 3.2 nM/min NO from xanthine while similar rates (22.9 ± 4.8) were observed with NADH. As O 2 tension increased to 2%, O 2 concentration-dependent delays in the onset of NO production occurred. Concurrent measurement of O 2 concentrations revealed that NO 2 - -dependent NO generation did not occur until all O 2 was consumed, suggesting that O 2 decreases NO 2 - reduction by indirect oxidation of the Mo via e − withdrawal at the FAD. Experiments with XDH, which has lower affinity for O 2 , did not demonstrate improved rates of NO formation or reduced lag times. However, immobilization of XO on artificial GAGs conferred the capacity for pronounced and sustained NO formation. In addition, reduction of NO 2 - by GAG-bound XO resulted not only in NO formation but concomitant diminution of XO-derived ROS. GAG-mediated enhancement of nitrite reductase activity and reduction in XO-derived ROS formation was confirmed when XO was associated with endothelial cell GAGs. Combined, these data indicate under inflammatory conditions where vascular XO levels and XO-GAG interactions are enhanced that O 2 -mediated inhibition of XO-catalyzed NO 2 - reduction can be overcome resulting in both increased NO generation and decreased ROS production.