EPR OF METHEMOGLOBIN FORMATION IN RED CELLS W/ UNSTABLE HEMOGLOBIN

衣藻叶绿体中的Hb已经被 克隆并在大肠杆菌中表达。这种HB类似于几种食草动物 在B10中有Tyr残基和E7位置中有过剩， 这两个分子都被认为参与了结合O2的氢键 这导致了极慢的折扣率。另一方面， 衣藻Hb的不同寻常之处在于，铁蛋白具有 高自旋到低自旋(六坐标)的转变pk为8.5，而 铁蛋白具有从高自旋到低自旋(六坐标)。 过渡PK为6.4。这表明Low的远端配体 铁蛋白和铁蛋白的自旋形式可能不同。 铁蛋白表现为单一的低自旋形式，其g值为 2.52、2.31、1.86，因此具有与肌红蛋白相似的各向异性 氢氧化物。然而，17O EPR和18O共振拉曼研究未能 提供氢氧化物配体的直接证据。光谱学研究 B10酪氨酸、E7谷氨酸和E10赖氨酸残基的远端突变 到目前为止还没有确定可能的氨基酸配体。进一步 正在进行研究，以确定远端配体和 了解不同寻常的低自旋EPR谱。

A Hb from the chloroplast of Chlamydomonas eugametos has been cloned andexpressed in E. coli. This Hb resembles several invetebrate in having a tyr residue in the B10 and a glut in the E7 positions, both of which are believed to participate in H-bonding to bound O2 that results in extremely slow off-rates. On the other hand, Chlamydomonas Hb is unusual in that the ferrous protein has a high-spin to low spin (six-coordinate) transition pK of 8.5, whereas the ferric protein has a high-spin to low spin (six-coordinate) transition pK of 6.4. This suggests that the distal ligand of the low spin forms of the ferrous and the ferric proteins may be different. The ferric protein exhibits a single low spin form with g values of 2.52, 2.31, 1.86, and thus an anisotropy similar to that of myoglobin hydroxide. However, 17O EPR and 18O resonance Raman studies failed to provide direct evidence for a hydroxide ligand. Spectroscopic studies on the distal mutants of the B10 Tyr, E7 Gln, and the E10 Lys residues have so far failed to identify a possible amino acid ligand. Further studies are being conducted to identify the distal ligand and to understand the unusual low spin EPR spectrum.