Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca 2؉ -dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophindeficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wildtype cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.cardiomyocytes ͉ cytochrome oxidase ͉ oxidative phosphorylation ͉ respiration ͉ chronoamperometry T he existence of a nitric oxide synthase (NOS) that is localized in the mitochondria (mtNOS) was originally described in a series of immunohistochemical studies published between 1995 (1, 2) and 1996 (3). Because all of the known NOS isoforms are encoded by nuclear DNA and NOS is not encoded by mitochondrial DNA, this finding implied that one of the recognized NOS isoforms was targeted to the mitochondria after protein synthesis in the cytosol. In these early studies, it was reported that the endothelial NOS (eNOS) isoform was localized to the inner mitochondrial membrane in all tissues that were tested, which included brain, kidney, liver, and skeletal and cardiac muscle. Between 1997 and 1998, more in-depth studies using a variety of NO detection techniques with isolated rat liver mitochondria (4), submitochondrial particles (SMPs; ref. 4), and purified NOS enzyme (5-7) added further support for the existence of a mtNOS. However, these studies were unable to determine whether the enzyme was novel or related to the neuronal (nNOS), inducible (iNOS), or eNOS isoforms. In the last three years, some laboratories have extended their studies to the investigation of the functional implications of mtNOS (8-11), while others have used a NO-sensitive dye to stain the mitochondria in intact cells and demonstrate the presence of NO within these organelles (12). Howeve...

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Although the function of metallothionein (MT), a 6-to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca 2؉ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT ؊͞؊ ) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed. Metallothioneins (MT) are 6-to 7-kDa intracellular cysteine-rich (30 mol%) metal binding proteins whose function remains elusive (1). A critical role for MT in protection against toxic non-essential metals such as cadmium is apparent (2), and MT appears to act as an antioxidant under a variety of conditions (3). More recently, in vitro data support the hypothesis that MT is a critical link between cellular redox state and metal ion homeostasis (4-6). In this regard, cysteines of metal thiolate clusters confer unique redox sensitivity to an otherwise redox inert metal ligand (e.g., zinc) and facilitate the potential for MT to participate in intracellular signal transduction pathways (7). In the current study, we examine this latter novel hypothesis in intact cells and tissue.We chose to study the interaction of MT and the free radical, nitric oxide (NO), because (i) the bioregulatory targets of NO usually contain cysteines and͞or metals at their active or allosteric site (8); (ii) NO (or a secondary product) reacts with MT in vitro, leading to the release of zinc (9) or cadmium (10); (iii) NO can form stable EPR-detectable complexes with MT in vitro (11); and (iv) MT can reduce the sensitivity of cells to potential toxic levels of NO (12). Application of a chimeric construct (called FRET-MT) based on a recently described cameleon for calmodulin (13) revealed an NO-induced conformational change in MT, indicative of metal release, thereby providing the first demonstrations of (i) changes in intramolecular FRET (fluorescence resonance energy transfer) of a native protein; and (ii) metal release from MT in response to physiologic stimuli in intact cells. Furthermore, the lack of myogen...