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...

Organisms rely on regulation at the molecular level, such as the allosteric regulation of hemoglobin (Hb) function by anions, to meet challenges presented by changing environmental and physiological conditions. A comparison of the effects of anions on oxygenation, oxidation, and sulfhydryl reactivity of Hb leads us to suggest that a large and significant part of the shift in oxygen affinity brought about by anion binding occurs as a result of increased conformational rigidity of the T state of deoxy Hb. As conformational rigidity increases, it becomes increasingly difficult for subunits in the deoxygenated T-state tetramer to assume higher oxygen affinity forms (T', T", T"'...) with less steric hindrance. The oxygen affinity reflects the average of the rapidly equilibrating conformations within the T state and is correspondingly decreased when anion levels are increased. The initial stage of the oxidation of Hb is relatively insensitive to steric alterations and thus reflects, primarily, the electronic aspects of the quaternary (T, T', T", T"'...) <--> equilibrium. We show that the reactivity of the sterically obscured sulfhydryl of beta93 Cys in deoxy Hb is much greater in chloride-free buffers than in buffers with added chloride. Anion-induced decreases in the extent and frequency of conformational fluctuations of subunits within the T-quaternary state thus reduce sulfhydryl reactivity as well as oxygen affinity. This parallel in anionic control of function allowed us to test, and disprove, the possibility that uncompensated positive charges in the central cavity of Hb Deer Lodge increase the frequency and extent of conformational fluctuations in its deoxy structure. This Hb variant exhibits increased anion sensitivity, increased oxygen affinity, and increased ease of oxidation, but without increased reactivity of its sulfhydryl groups, indicating that active-site alterations in deoxy Hb Deer Lodge are primarily electronic and not associated with increased conformational fluctuations within its T state. The restoration of normal properties in Hb Deer Lodge by addition of anions reinforces our conclusion that anionic control can be exerted through both steric and electronic alterations. The anionic control of fluctuations within the T state of Hb illustrates an important principle of macromolecular structure-function relationships: that functional regulation can be achieved by alterations in conformational rigidity.

Previous studies showed that CO/H 2 O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb ␤ chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/ H 2 O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-␤93 with Nethylmaleimide. Furthermore, this internal electrontransfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (␤2His 3 Arg) and other species lacking His-␤2 than in Hb A 0 . This difference is consistent with preferential binding of Cu(II) in Hb A 0 to a high affinity site involving His-␤2, which is ineffective in promoting electron exchange between Cu(II) and the ␤ heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R 7 T conformational equilibrium. The ␤ hemes in Cu(II)-metHb are reduced under CO at rates close to those observed for cytochrome c oxidase, where heme and copper are present together in the oxygen-binding site and where internal electron transfer also occurs.

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