Protein Design Laboratory, Yokohama City University* Faculty of Engineering, Hosei University, Japan** MRC Laboratory of Molecular Biology, UK***
○Satoru Unzai* Kiyohiro Imai** Sam-Yong Park* Jeremy Tame* Kiyoshi Nagai***
Blood from the trout Oncorhyncus mykiss (formerly Salmo irideus) contains four distinct hemoglobins (Hbs). Trout Hb IV makes up roughly 65% of the total Hb component and it is characterized by the Root effect, an unusually strong dependence of oxygen affinity on pH. Below pH 6 this Hb binds oxygen weakly and non-cooperatively. This allows discharge of oxygen into the swim bladder by the secretion of lactic acid into the blood in the gas gland.
In addition to protons, intracellular Hb function is also mediated by organic phosphates. Erythrocytes of teleost fish use ATP, GTP, or IHP (inositol hexaphosphate) as allosteric effectors. Addition of ATP tends to decrease both the overall oxygen affinity and the cooperativity of trout Hb IV.
Here we report the crystal structure of met form trout Hb IV bound to ATP, refined to 1.70 A resolution. Electron density corresponding to ATP was found in the cleft between the two beta chains. Although the trout Hb IV was met form, its quaternary structure was very similar to that of deoxy (T state) trout Hb I, another trout Hb component. Because the crystal was formed at low pH (pH 5.5) and in the presence of ATP it appears that the T state was strongly stabilized. The position of the ATP molecule in the trout Hb IV central cavity is deeper than that of DPG molecule in the deoxy human Hb A. In fish Hbs, the Asp residue in position beta-NA2 was suggested to form contacts with ATP, however, the side chain points into solvent and does not make contact with ATP or other amino acid residues in the trout Hb IV structure.