Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.

S-Nitrosated hemoglobin (SNO-Hb) is of interest because of the allosteric control of NO delivery from SNO-Hb made possible by the conformational differences between the R-and T-states of Hb. To better understand SNO-Hb, the oxygen binding properties of Snitrosated forms of normal and sickle cell Hb were investigated. Spectral assays and electrospray ionization mass spectrometry were used to quantify the degree of S-nitrosation. Hb A 0 and unpolymerized Hb S exhibit similar shifts toward their R-state conformations in response to S-nitrosation, with increased oxygen affinity and decreased cooperativity. Responses to 2,3-diphosphoglycerate were unaltered, indicating regional changes in the deoxy structure of SNO-Hb that accommodate NO adduction. A cycle of deoxygenation/ reoxygenation does not cause loss of NO or appreciable heme oxidation. There is, however, appreciable loss of NO and heme oxidation when oxygen-binding experiments are carried out in the presence of glutathione. These results indicate that the in vivo stability of SNO-Hb and its associated vasoactivity depend on the abundance of thiols and other factors that influence transnitrosation reactions. The increased oxygen affinity and R-state character that result from S-nitrosation of Hb S would be expected to decrease its polymerization and thereby lessen the associated symptoms of sickle cell disease. Hemoglobin (Hb)1 is of central importance to human health in its role as a respiratory protein. Another chapter in the study of the human health significance of Hb is beginning, focused on NO uptake and delivery by Hb and the role this plays in the control of blood pressure and other NO-dependent reactions. Nitrosation of sulfhydryl groups on the Hb tetramer creates S-nitrosated Hb (SNO-Hb), which has been shown to play an important role in NO uptake and delivery (1). S-Nitrosated forms of proteins such as Hb can be formed via interaction with nitrosating agents formed upon interaction of NO and oxygen (NO x ) and by NO-exchange reactions (transnitrosations) with nitrosated forms of low molecular weight thiols such as cysteine and glutathione. Conversely, the low molecular weight thiols can act as NO acceptors in transnitrosation reactions where NO is donated by S-nitrosated proteins (2, 3).Hb-based NO transport via SNO-Hb is significant because it can greatly extend the range of NO-dependent reactions. Unlike SNO-Hb, free NO is a very reactive molecule, whose lifetime in the complex cellular milieu would be expected to be very short. It is this characteristic of NO that delayed the discovery of NO-dependent reactions in smooth muscle relaxation, platelet inhibition, neurotransmission, and immune regulation (4 -8). What is learned about Hb-based NO transport will have far-ranging applications in these disparate fields.The studies reported here concern the oxygen binding properties of variably S-nitrosated adult human hemoglobin (Hb A 0 ) and sickle cell hemoglobin (Hb S) that has a Glu 3 Val substitution at ␤6. Although physiological levels of S-nitrosa...

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