An additional H-bond in the α1β2 interface as the structural basis for the low oxygen affinity and high cooperativity of a novel recombinant hemoglobin (βL105W)

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/beta L105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha (1)/beta (2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P-50 = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P-50 = 9.9 mmHg, n(max) = 3.2 at pH 7.4), rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(beta L105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha (1)/beta (2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on N-15-labeled rHb(beta L105W) have identified the indole nitrogen-attached H-1 resonance of beta 105Trp for rHb(beta L105W). H-1 NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O-2 affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha (1)/beta (2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O-2 affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.