Under such “standardized” conditions (similar reducing power and nitrite and enzyme concentrations), the amount of NO formed by XO, XD and AO is similar, with the significance of XOR-derived NO formation does not depend on the XOR form present, XD or XO (note that the XOR predominant form present under different physiological and pathological conditions is still a matter of debate). Open in a separate window Fig. the current knowledge on two of those “non-dedicated nitrite reductases”, the molybdoenzymes xanthine oxidoreductase and aldehyde oxidase, discussing the and studies to provide the current picture of the role of these enzymes on the NO metabolism in humans. oxidase; DAF-FM, 4-amino-5-methylamino-2,7-difluorofluorescein; DAF-FM DA, DAF-FM diacetate; DPI, diphenyleneiodonium chloride; EPR, electron paramagnetic resonance; Hb, hemoglobin; HepG2, human epithelial cells from liver carcinoma; HL, human liver; HMEC, human microvascular Biperiden endothelial cells; L-NAME, N-nitro-L-arginine methyl ester hydrochloride; mARC, mitochondrial amidoxime reducing component; Mb, myoglobin; MGD2-Fe, iron-oxidase [31] or dioxygen [32], [33], [34]). Open in a separate window Fig. 2 “Classic” and novel pathways of NO formation. The “classic” pathways of NO formation (black arrows, grey shadowed area) are catalyzed by NOS, complex homodimeric enzymes, constituted by one flavinic reductase heme iron of the oxygenase domain; on the heme, the dioxygen is definitely triggered to hydroxylate L-arginine; the N-hydroxy-L-arginine created is definitely, then, oxidized to yield L-citrulline and NO. To control the specificity of NO signaling (indigo arrows and text), and also to limit the NO toxicity, NOS are tightly controlled and the NO life time is definitely controlled through its quick oxidation to nitrate and nitrite. The novel Biperiden pathways of NO formation (violet arrows and text) are reductive in nature (contrary to the oxidative NOS-catalyzed pathways) and are dependent on the nitrite reduction under hypoxic and anoxic conditions. These pathways are catalyzed by “non-dedicated nitrite reductases”, metalloproteins that are present in cells to perform additional functions, including several heme proteins and molybdoenzymes. The NO biological effects are accomplished (green arrows and text), primarily, by post-translational changes of cysteine residues and additional thiols and of transition metal centers, mostly labile [4Fe-4S] centers and hemes (as is the case of the well known activation of guanylate cyclase), to yield nitrosothiol (-S-N=O) and nitrosyl (-metal-N=O) derivates, respectively. 3.?Nitrite-derived NO At the same time as our knowledge about the physiological roles of NO in human beings was growing exponentially, nitrate and nitrite were overlooked and considered “ineffective” end-products of NO metabolism. This dogma changed in the early XXI century, when it became obvious that nitrite can be reduced back to NO under hypoxic conditions (Eq. (1)) and it was re-discovered that nitrite administration can be cytoprotective during ischemia and additional pathological conditions (Fig. 2) [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69] ((Cc) [75], cytochrome P450 [76], cytochrome oxidase (CcO) [77], [78], [79], and several additional proteins [80], [81], [82], [83]. or observed nitrite effects with the knowledge of nitrite reduction through those varied pathways? (iv) How are all those pathways orchestrated and Biperiden studies to provide the best possible current picture of the role of these enzymes within the NO rate of metabolism. 5.?Human being XOR and AO XOR is definitely a key enzyme in purine catabolism, where it catalyzes the oxidation of both hypoxanthine and xanthine to the terminal metabolite, urate [97], [98], [99], [100], [101], [102]. AO catalyzes the oxidation of aldehydes into the respective carboxylates and, although its physiological function remains a matter of argument, it seems to be a probable partner in the rate of metabolism of some neurotransmitters and retinoic acid [103], [104], [105], [106], [107], [108]. Both enzymes contribute also to the xenobiotic rate of metabolism (because of the low substrate specificity) and are allegedly involved in signaling (physiological conditions) and oxidative stress-mediated pathological conditions (because of the Biperiden ability to form reactive oxygen varieties, superoxide anion radical and hydrogen peroxide) [109], [110], [111], [112], [113], [114], [115], [116], [117], Fgfr2 [118], [119], [120], [121], [122], [123], [124], [125], [126], [127], [128], [129], [130], [131], [132], [133], [134], [135], [136], [137], [138], [139], [140], [141], [142], [143], [144]. Ag/AgCl) enclosed by a gases-only-permeable membrane (that ensures the electrochemical measurements selectivity). That NO is the product of XOR and AO-catalyzed nitrite reduction was unequivocally shown by EPR, using the spin-trap iron-?2.04, having a hyperfine splitting of 1 1.27mT. Data adapted with permission from Ref.?[137]. Copyright 2015 American Chemical Society. (C) Assays with the NO-selective electrode. The kinetic mechanism type, “ping-pong”, is definitely schematically indicated within the remaining panel. In both methodologies, the gray lines refer to curves without enzyme; black collection, without nitrite; red and dark.