Protonation states of buried histidine residues in human deoxyhemoglobin revealed by neutron crystallography

中性子結晶構造解析により解明されたヒト・デオキシヘモグロビンにおける埋没ヒスチジン残基のプロトン化状態

茶竹 俊行*; 柴山 修哉*; Park, S. Y.*; 栗原 和男; 玉田 太郎; 田中 伊知朗*; 新村 信雄*; 黒木 良太; 森本 幸生*

Chatake, Toshiyuki*; Shibayama, Naoya*; Park, S. Y.*; Kurihara, Kazuo; Tamada, Taro; Tanaka, Ichiro*; Niimura, Nobuo*; Kuroki, Ryota; Morimoto, Yukio*

A large crystal of human deoxy hemoglobin (Hb) was grown from DO solution (pD 6.3). The preliminary neutron diffraction experiment was carried out at the KUR reactor in RRI of Kyoto University, and the diffraction data set to 2.1 resolution was collected at JRR-3 reactor in JAEA using the BIX-3. The neutron crystal structure of Hb reveals that both the - and -distal histidines (His58 and His63) adopt fully (doubly) protonated form. This finding sharply contrasts with existing results on R (relaxed) state liganded Hbs where such full protonation can never occur. This results suggest an interesting possibility that the both histidines could contribute to the T (tense) state Bohr effect of Hb. Indeed, the protonation/deprotonation of each distal histidine may have a direct impact on the oxygen affinity of the nearby heme group through sterical hindrance and/or polarity change in the heme pocket without affecting the allosteric equilibrium of Hb.