The linkage between the proximal histidines and the proximal polypeptide in normal adult human hemoglobin (Hb A) has been proposed to play a major role in transmitting allosteric effects between oxygen binding sites [Perutz, M. F. (1970) Nature 228, 726-734]. Here we present circular dichroism (CD), H-1 NMR, analytical ultracentrifugation, and stopped-flow kinetic data to better define the quaternary structure of hemoglobins in which the linkage between the proximal histidines and the polypeptide backbone has been broken [Barrick et al. Not. Struct. Biol. 4, 78-83 (1997)] and to characterize the distal ligand binding properties of these proximally detached Hbs. CD spectroscopy indicates that rHb (alpha H87G) and rHb (alpha H87G/beta H92G) retain at least partial T-quaternary structure with distal ligand bound, whereas rHb (beta H92G) does not, consistent with H-1 NMR spectra. Analytical ultracentrifugation reveals significant tetramer dissociation in rHb (beta H92G) to be the likely cause of loss of T-state markers. These quaternary structure studies indicate that in distally liganded Hb, the T-state is compatible with proximal linkages in the beta- but not the alpha -chains. H-1 NMR titrations of rHb (alpha H87G) with n-butyl isocyanide demonstrate the alpha -chains to be of high affinity as compared with the beta -chains. Comparing Ligand association and dissociation rates between the rHb (alpha H87G) variant with the T- and R-states of wild-type Hb A indicates that at the alpha -chains, carbon monoxide affinity is modulated entirely by the proximal Linkage, rather than from distal interactions. Some residual allosteric interactions may remain operative at the beta -chains of rHb (alpha H87G).