Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.

Rate constants for hemin dissociation from the ␣ and ␤ subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37°C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated ␣ and ␤ chains and for recombinant hemoglobin tetramers stabilized by ␣ subunit fusion. The rate constant for hemin dissociation from ␤ subunits in native hemoglobin increases from 1.5 h ؊1 in tetramers at high protein concentration to 15 h ؊1 in dimers at low concentrations. The rate of hemin dissociation from ␣ subunits in native hemoglobin is significantly smaller (0.3-0.6 h ؊1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-␣ subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric ␣ and ␤ chains are much greater, 12 and 40 h ؊1 , respectively, at pH 7, 37°C. Aggregation of monomers to form ␣ 1 ␤ 1 dimers greatly stabilizes bound hemin in ␣ chains, decreasing its rate of hemin loss ϳ20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to ␤ subunits. A significant reduction in the rate of hemin loss from ␤ subunits does occur after formation of the ␣ 1 ␤ 2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.

The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins. A hydrogen bond between the N epsilon proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides. To test these ideas, engineered mutants of sperm whale myoglobin and the alpha- and beta-subunits of human haemoglobin were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the alpha-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to beta-chains within R-state haemoglobin. In the beta-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.

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