The pulmonary vasculature, particularly the endothelium, is known to be a key site responsive to both physiological and pathological changes in 02 delivery and exposure to endogenous NO. Mitochondria are critical loci of cellular respiration, biosynthesis, and metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS). We have established that mitochondrial respiratory complexes I (NADH dehydrogenase) and Ill (cytochrome c reductase) are primary locations of irreversible inhibition by RNS and that complex IV (cytochrome c oxidase), in addition to its known cytochrome c:O2 oxidoreductase activity, may also function as an NO oxidase. Accordingly, we hypothesize that under normal physiological circumstances, the NO oxidase activity of complex IV limits formation of RNS by rapidly converting NO to the relatively innocuous nitrite ion. The efficiency of this catabolic reaction, which also consumes 02, depends upon the prevailing NO/O2 ratio. Under hyperoxic conditions, or in pathophysiological circumstances where the electron-transport chain has been compromised, RNS (and ROS) formation is not effectively suppressed. This results in irreversible inhibition of complexes I and III, which in turn exacerbates the situation by increasing the production of the damaging reactive species and ultimately, leads to cell death. In testing this broad hypothesis, we propose to determine: 1) that the NO oxidase activity of complex IV limits nitrosative stress in pulmonary endothelial cells; 2) the molecular sites at which RNS irreversibly inhibit complex I and complex III; 3) the extent to which nitrosative stress may affect the interaction of cytochrome c with complex Ill and/or complex IV; 4) the functional role of RNS-dependent modification of the interaction between cytochrome c and complexes III and/or IV in promoting the loss of cytochrome c from the intermembrane space during proapoptotic stimulation of cultured pulmonary endothelial cells.
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