Search

S-Nitrosated hemoglobin is remarkably stable and can be cycled between deoxy, oxygenated, or oxidized forms without significant loss of NO. Here we show thatS-nitrosation of adult human hemoglobin (Hb A0) or sickle cell Hb (Hb S) results in an increased ease of anaerobic heme oxidation, while anions cause redox shifts in the opposite direction. The negatively charged groups of the cytoplasmic domain of Band 3 protein also produce an allosteric effect on S-nitrosated Hb. Formation and deoxygenation of a SNO-Hb/Band 3 protein assembly does not in itself cause NO release, even in the presence of glutathione; however, this assembly may play a role in the migration of NO from the red blood cells to other targets and may be linked to Heinz body formation. Studies of the anaerobic oxidation of Hb S revealed an altered redox potential relative to Hb A0that favors met-Hb formation and may therefore underlie the increased rate of autoxidation of Hb S under aerobic conditions, the increased formation of Heinz bodies in sickle cells, and the decreased lifetime of red cells containing Hb S. A model for the interrelationships between the deoxy, oxy, and met forms of Hb A0and Hb S, and their S-nitrosated counterparts, is presented.

The Fe3+binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe3+binding constant in the presence of 350 μmphosphate at pH 6.5 and 25 °C was determined as 2.4 × 1018m−1. EPR spectra for the recombinant Fe3+nFbp gave g′ = 4.3 and 9 signals characteristic of high spin Fe3+in a strong ligand field of low (orthorhombic) symmetry. 31P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe3+in apo-nFbp. Finally, an uncorrected Fe3+/2+redox potential for Fe-nFbp was determined to be −290 mV (NHE) at pH 6.5, 20 °C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe3+binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria.

Previous studies showed that CO/H2O oxidation provides electrons to drive the reduction of oxidized hemoglobin (metHb). We report here that Cu(II) addition accelerates the rate of metHb beta chain reduction by CO by a factor of about 1000. A mechanism whereby electron transfer occurs via an internal pathway coupling CO/H2O oxidation to Fe(III) and Cu(II) reduction is suggested by the observation that the copper-induced rate enhancement is inhibited by blocking Cys-beta93 with N-ethylmaleimide. Furthermore, this internal electron-transfer pathway is more readily established at low Cu(II) concentrations in Hb Deer Lodge (beta2His --> Arg) and other species lacking His-beta2 than in Hb A0. This difference is consistent with preferential binding of Cu(II) in Hb A0 to a high affinity site involving His-beta2, which is ineffective in promoting electron exchange between Cu(II) and the beta heme iron. Effective electron transfer is thus affected by Hb type but is not governed by the R left arrow over right arrow T conformational equilibrium. The beta 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.