Our studies constituted the first ones providing evidence for a production of nitric oxide by purified mitochondria. The production of this molecule was demonstrated by the oxidation of oxymyoglobin, electron spin resonance spectroscopy with the spin trapping technique, and L-citrulline production. These techniques along with the use of purified, intact mitochondria (from rat liver or from hepatocytes) provided experimental support for a production of nitric oxide by these organelles. Further evidence was furnished by the isolation of a mitochondrial nitric-oxide synthase with characteristics to other isoforms. The production of nitric oxide by mitochondria modulated the oxygen consumption and ATP production of mitochondria. This was accomplished through the competitive inhibition of cytochrome oxidase. Thus, nitric oxide exhibits effects not necessarily mediated via the activation of the soluble guanylate cyclase, pointing at a different target site, in this case, cytochrome oxidase. Given that this production of nitric oxide may establish the basis for a novel regulatory pathway--considering the ubiquitous distribution of mitochondria in tissues and the relevant role this molecule may have on energy metabolism, oxygen consumption, and oxygen free radical production--, it becomes imperative to study the biochemistry of the enzyme responsible for this production in mitochondria. This proposal is aimed at studying the regulation of the enzyme by posttranslational modifications and how these modifications alter or modulate the targeting of the enzyme and/or its activity. Studying the biochemistry of mtNOS will provide a better understanding of the role of nitric oxide in the cell-mitochondria interactions relevant to physiological situations.