Abstract: Nitric oxide (NO) is produced by one of three mammalian nitric oxide synthase enzymes (NOS) and is a key signaling molecule, which affects physiologies such as smooth muscle relaxation and neurotransmission. These processes are mediated via NO binding to its heme target in soluble guanylate cyclase (sGC). Upon binding, the second messenger cyclic guanosine monophosphate (cGMP) is rapidly synthesized. NO signaling in this manner is well established; however, there is an increasing role for cGMP-independent NO signaling. While key molecular details remain unanswered, S-nitrosation represents an example of cGMP-independent NO signaling. This modification has garnered recent attention as it has been shown to modulate the function of several important biochemical pathways, including apoptosis. Although an analogy to O-phosphorylation can be drawn, little is known about protein nitrosothiol regulation in vivo. It was known for over a decade that NO inhibits the further production of NO through an unknown mechanism. We can now attribute at least part of this loss in NOS activity to S-nitrosation of the zinc tetrathiolate cluster of NOS. Work in this thesis provides both in vitro and in vivo evidence that this is a physiologically relevant regulatory mechanism. A greater understanding of how S-nitrosation is mediated has broad implications for cGMP-independent signaling. Other chapters in this thesis focus on the NO-dependent inhibition of apoptosis, which is another observation that can be explained by S-nitrosation. While working on the apoptotic cysteine protease, caspase-3, it was determined that free NO and a biological transnitrosating agent (S-nitrosoglutathione, GSNO) react with four of the eight cysteines in caspase-3. The same four cysteines were found to be S-nitrosated even when using a modest concentration of S-nitrosating agent, which exemplifies the promiscuous reactivity of free NO and small molecule nitrosothiols. Previous in vivo work has demonstrated that caspase-3 is S-nitrosated solely on the catalytic cysteine, indicating that formation of caspase-3-SNO may be protein-assisted. Using purified enzymes, we demonstrated that a single cysteine in thioredoxin-1 is capable of a targeted, reversible transnitrosation reaction with only the catalytic cysteine of caspase-3. This process proceeded two orders of magnitude faster than both a NO donor and GSNO and rapidly resulted in catalytically inactive caspase-3. Additionally, this transnitrosation reaction was found to occur in cultured human T-cells and suggests a new anti-apoptotic role for thioredoxin.