Hemoglobin Kirklareli (α H58L), a New Variant Associated with Iron Deficiency and Increased CO Binding

Mutations in hemoglobin can cause a wide range of phenotypic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin alpha chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) -> Leu mutation in alpha 1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (similar to 16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the alpha H58L replacement. Mutant alpha subunits containing Leu-58(E7) autoxidize similar to 8 times and lose hemin similar to 200 times more rapidly than native alpha subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the alpha H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O-2, promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant alpha subunit has an similar to 80,000-fold higher affinity for CO than O-2, causing it to rapidly take up and retain carbon monoxide, which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.