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An additional H-bond in the alpha 1 beta 2 interface as the structural basis for the low oxygen affinity and high

T Y Fang1, V Simplaceanu, C H Tsai

  • 1Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.

Biochemistry
|November 15, 2000
PubMed

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), 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(betaL 105W) 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 (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H 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.

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