Abstract-Previous studies have revealed a novel interaction between deoxyhemoglobin and nitrite to generate nitric oxide (NO) in blood. It has been proposed that nitrite acts as an endocrine reservoir of NO and contributes to hypoxic vasodilation and signaling. Here, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 36 times faster than deoxyhemoglobin because of its lower heme redox potential. We hypothesize that physiologically this reaction releases NO in proximity to mitochondria and regulates respiration through cytochrome c oxidase. Spectrophotometric and chemiluminescent measurements show that the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant of 12.4 mol/L -1 s -1 (pH 7.4, 37°C). Because the IC 50 for NO-dependent inhibition of mitochondrial respiration is approximately 100 nmol/L at physiological oxygen tensions (5 to 10 mol/L); we tested whether the myoglobin-dependent reduction of nitrite could inhibit respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate containing both myoglobin and mitochondria resulted in NO generation and inhibition of respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by the xanthine oxidoreductase inhibitor allopurinol. These data expand on the paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to regulate NO-dependent signaling. (Circ Res. 2007;100:654-661.)

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three "codons," into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.Nature generates functional biological molecules by subjecting libraries of nucleic acids to iterated cycles of translation, selection, amplification, and diversification (1-4). Compared with analogous synthesis and screening methods currently used to discover synthetic molecules with desired properties, these evolution-based approaches are attractive because of the much larger numbers of molecules that can be simultaneously evaluated, the minute quantities of material needed, and the relatively modest infrastructure requirements for library synthesis and processing (1-4).Despite these attractions, evolution-based approaches can only be applied to molecules that can be translated from amplifiable information carriers. We previously described the generality of DNA-templated organic synthesis (DTS) and explored its potential for translating DNA sequences into corresponding synthetic products by using DNA hybridization to modulate the effective molarity of DNA-linked reactants (5). DTS can generate products unrelated in structure to the DNA backbone in a sequence-specific manner (5,6), does not require functional group adjacency to proceed efficiently (5,7), can mediate sequence-programmed multistep small-molecule synthesis (8), and can enable reaction pathways that are difficult or impossible to realize with the use of conventional synthetic strategies (9).These features of DTS raise the possibility of translating single-solution libraries of DNA sequences into corresponding libraries of synthetic small molecules conjugated to their respective templates. Because each member of a DNA-templated synthetic library is linked to an encoding nucleic acid, these libraries are suitable for in vitro selection (10), polymerase chain reaction (PCR) amplification, and DNA sequence characterization to reveal the identity of synthetic library members possessing functional properties (Fig. 1) integration of these concepts into the DNA-templated synthesis of a library of macrocycles ( Fig. 2A), the selection of this pilot library for affinity to a target protein, and the identification of a functional library member through the amplification and characterization of DNA sequences surviving the selection.Although macrocycles can be cha...

Nitrite reacts with deoxyhemoglobin to form nitric oxide (NO) and methemoglobin. Though this reaction is experimentally associated with NO generation and vasodilation, kinetic analysis suggests that NO should not be able to escape inactivation in the erythrocyte. We have discovered that products of the nitrite-hemoglobin reaction generate dinitrogen trioxide (N2O3) via a novel reaction of NO and nitrite-bound methemoglobin. The oxygen-bound form of nitrite-methemoglobin shows a degree of ferrous nitrogen dioxide (Fe(II)-NO2*) character, so it may rapidly react with NO to form N2O3. N2O3 partitions in lipid, homolyzes to NO and readily nitrosates thiols, all of which are common pathways for NO escape from the erythrocyte. These results reveal a fundamental heme globin- and nitrite-catalyzed chemical reaction pathway to N2O3, NO and S-nitrosothiol that could form the basis of in vivo nitrite-dependent signaling. Because the reaction redox-cycles (that is, regenerates ferrous heme) and the nitrite-methemoglobin intermediate is not observable by electron paramagnetic resonance spectroscopy, this reaction has been 'invisible' to experimentalists over the last 100 years.