The electrostatic theory for ligand discrimination in myoglobin is based on the physiological importance of hydrogen bonding between the distal histidine and bound ligands in myoglobin. A quantitative estimation of this theory in hemoglobin had not been established. A series of 'electrostatic' mutations was created to examine for the existence of electrostatic stabilization of bound O2 in both the alpha and beta subunit of R-state recombinant human hemoglobin. A set of HisE7 to Ala, Leu, and Gln replacements was used to investigate the role of the distal histidine. The aliphatic mutations, AlaE7 and LeuE7, abolish all hydrogen bonding capabilities near bound ligands and cause dramatic increases in ligand association rate constants, particularly for O2. The GlnE7 substitution retains hydrogen bonding capabilities and results in wild-type-like ligand binding behavior. A second set of mutations were created to alter and examine the electrostatic potential in the distal pocket: PheCD4 → Val, ValE11 → Asn, and the double mutant HisE7 → Leu/ValE11 → Asn. The ValCD4 mutation increases the flexibility of the distal histidine side chain, weakening hydrogen bonding interactions with bound ligands. The addition of an asparagine into the distal pocket at position E11 provides a second hydrogen bonding donor in both subunits. The LeuE7/AsnE11 double mutation maintains 'wild-type' electrostatic behavior, due to the compensatory effects of the addition of a hydrogen bond donor at position E11 and removal of one at position E7. Finally, the O2 and CO properties of the double mutant HisE7 → Gln/LeuB10 → Trp were examined as a blood substitute prototype with reduced rates of ligand capture but normal or increased rates of ligand dissociation. This combination of amino acid replacements causes marked decreases in O2 affinity in both subunits and maintains high rates of dissociation, both of which are favorable for efficient O2 transport. The rate of association is decreased by the large size of the Tip side chain and the weakening of hydrogen bonding by the GlnE7 replacement markedly enhances O2 dissociation compared to that measured for the single TrpB10 mutants.