The discovery of superoxide dis-mutase by McCord and Fridov-ich (1) ushered in a new area of biology wherein free radicals had to be factored into the biology of animals and plants. The free radical species of oxygen, the superoxide anion radical, has become the focus of thousands of studies. In medicine, the number of disease processes that have been linked to overexposure to oxidants or a failure to defend against them is vast, including pathologies as diverse as neurodegeneration, sepsis, atherosclerosis, and arthritis. The NO free radical and its redox partners, the nitrosonium and nitroxide ions, have been similarly painted with broad negative brushes (2). More recently, NO has been recognized for its role in normal physiology, arising in part from its ability to act as a signal through regulation of guanylate cyclase (3) and to S-nitrosylate cysteinyl residues of proteins and peptides (3). In a recent issue of PNAS, we witnessed the emergence of a new paradigm wherein the interplay between different highly reactive species allows for complex and fast regulation of cellular processes whose disruption has potentially serious pathological consequences (4). Research from the Hare laboratory (5) has shown that the different isoforms of NO synthase (NOS) are involved in controlling different aspects of cardiac contractility. However, Khan et al.(4) present simple and clear studies that describe a new aspect to the paradigm of NO control that has broad application beyond the realm of contractility. Khan et al. show that the neuronal isoform of NOS (nNOS) and the superoxide-generating enzyme xanthine oxidoreductase (XOR) are in physical proximity in the sarcoplasmic reticulum (SR) of the cardiac myocytes of mice. Earlier studies have shown that superoxide production within cardiac myocytes has a potentially important signaling role (6). Furthermore, disruption of this signaling is involved in the development of cardiac pathologies such as congestive heart failure. Other research has also elucidated a key role for nNOS-generated NO in controlling cardiac contractility through altered intracellular calcium storage (7), potentially through the formation of SNO on the ryanodine receptor (RyR)(8). Through the elegant use of knockout mice, Khan et al.(4) have connected these observations, thereby demonstrating that the regulation of XOR-generated superoxide represents another site of NO modulation of cardiac contractility. This is an immediately attractive proposal, because NO and superoxide are both free radical species that have long been known to react at nearly diffusion-limited rates (9). For background, it is of value to briefly summarize the XOR system. XOR is widely distributed and is implicated in apoptosis and pathophysiology (8). In vivo, the system exists in two forms: xanthine dehydrogenase (XDH) and xanthine oxidase (XO). Both forms convert hypoxanthine to xanthine or xanthine to uric acid. However, XDH utilizes NAD+ as the electron acceptor, whereas XO transfers electrons to molecular oxygen, forming superoxide (10). XDH can be converted to XO either irreversibly by proteolytic cleavage or reversibly by thiol modification (11, 12). Importantly, XOR does not appear to associate with the other isoforms of NOS, particularly endothelial NOS (eNOS). Furthermore, within heart muscle both nNOS and XOR are found within the SR along with the calcium pump SERCA2a and the calcium channel RyR. Impressively, xanthine-mediated production of superoxide is significantly increased in nNOS knockout mice compared with wild-type and eNOS knockout animals. This enhanced superoxide production is not caused by altered XOR expression, nor is it …