Shiro Y, Iwata T, Makino R, Fujii M, Isogai Y, Iizuka T
Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
J Biol Chem. 1993 Sep 25;268(27):19983-90.
Tetrazole-myoglobin (Tet-Mb), a site selectively modified myoglobin with tetrazole anion (-CN4-) covalently attached to the imidazole N epsilon of the distal histidine 64(E7) (see Fig. 1; Kamiya, N., Shiro, Y., Iwata, T., Iizuka, T., and Iwasaki, H. (1991) J. Am. Chem. Soc. 113, 1826-1829), exhibited unique properties in the reduction from ferric to ferrous states and in the reaction of its deoxy form with O2. The redox potential of Tet-Mb is obtained to be -193 mV, which is much lower than that of unmodified (native) myoglobin (50 mV), possibly due to the electrostatic interaction between the heme iron and the tetrazole group. The ferrous deoxy form of Tet-Mb was rapidly oxidized to its ferric form in the reaction with O2 at room temperature through an intermediary formation of its oxy form and with the generation of O2-. The oxy form of Tet-Mb can be detected by the optical spectral measurement at -12 degrees C, the rapid scan measurement at room temperature, and the electron spin resonance measurement of its cobalt-substituted derivative (Tet-Mb(Co2+)) at 77 K. In the kinetic measurement of the O2 binding reaction to Tet-Mb, its association and dissociation rate constants in the bimolecular reaction were 6.1 x 10(7) M-1 s-1 and 2200 s-1, respectively, showing that the tetrazole modification of His-64 extremely accelerates its association and dissociation rates. Taken together with the extremely fast autoxidation rate (53 h-1) obtained, these kinetic results suggested that the channel of O2 from the solvent region to the protein interior is open enough to pass the external ligand. The structure is discussed in relation to those of some genetic mutants. Taking these properties, we demonstrated that Tet-Mb can catalyze O2 consumption to generate O2-, coupled with the NADH-supported enzymatic reduction system of cytochrome P-450cam under an aerobic condition.
四唑肌红蛋白（Tet-Mb）是一种位点选择性修饰的肌红蛋白，四唑阴离子（-CN4-）共价连接到远端组氨酸64（E7）的咪唑Nε上（见图1；神谷，N.，史郎，Y.，岩田，T.，饭冢，T.，和岩崎，H.（1991年）《美国化学会志》113，1826 - 1829），在从三价铁还原为二价铁状态以及其脱氧形式与O2的反应中表现出独特的性质。Tet-Mb的氧化还原电位为 -193 mV，远低于未修饰（天然）肌红蛋白的氧化还原电位（50 mV），这可能是由于血红素铁与四唑基团之间的静电相互作用。在室温下，Tet-Mb的亚铁脱氧形式在与O2的反应中通过其氧合形式的中间形成和O2-的生成迅速氧化为其三价铁形式。Tet-Mb的氧合形式可以通过在 -12℃下的光谱测量、室温下的快速扫描测量以及其钴取代衍生物（Tet-Mb（Co2+））在77 K下的电子自旋共振测量来检测。在对Tet-Mb的O2结合反应的动力学测量中，其二分子反应中的缔合和解离速率常数分别为6.1×10(7) M-1 s-1和2200 s-1，表明对His-64的四唑修饰极大地加速了其缔合和解离速率。结合所获得的极快的自氧化速率（53 h-1），这些动力学结果表明从溶剂区域到蛋白质内部的O2通道足够开放以通过外部配体。结合一些基因变体的结构对该结构进行了讨论。基于这些性质，我们证明了Tet-Mb可以在有氧条件下催化O2消耗以生成O2-，并与细胞色素P - 450cam的NADH支持的酶促还原系统偶联。
