Hirota Shun, Azuma Kayo, Fukuba Makoto, Kuroiwa Shigeki, Funasaki Noriaki
Department of Physical Chemistry, 21st Century COE Program, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan.
Biochemistry. 2005 Aug 2;44(30):10322-7. doi: 10.1021/bi0507581.
Human myoglobin (Mb) possesses a unique cysteine (Cys110), whereas other mammalian Mbs do not. To investigate the effect of a cysteine residue on Mb, we introduced cysteine to various sites on the surface of sperm whale Mb (K56C, V66C, K96C, K102C, A125C, and A144C) by mutation. The cysteines were inserted near the end of alpha-helices, except for V66C, where the cysteine was introduced in the middle of an alpha-helix. Reduction of the heme was observed for each mutant metMb by incubation at 37 degrees C under carbon monoxide atmosphere, which was much faster than reduction of wild-type metMb under the same condition. Heme reduction did not occur significantly under nitrogen or oxygen atmospheres. The rate constant for heme reduction increased for higher mutant Mb concentration, whereas it did not change significantly when the CO concentration was reduced from 100% CO to 50% CO with 50% O(2). The similarity in the rate constants with different CO concentrations indicates that CO stabilizes the reduced heme by coordination to the heme iron. SDS-PAGE analysis showed that mutant Mb dimers were formed by incubation under CO atmosphere but not under air. These dimers were converted back to Mb monomers by an addition of 2-mercaptoethanol, which showed formation of a Mb dimer through a disulfide bond. The rate constant decreased in general as the heme-cysteine distance was increased, although V66C Mb exhibited a very small rate constant. Since V66 is placed in the middle of an alpha-helix, steric hindrance would occur and prevent formation of a dimer when the cysteine residues of two different V66C Mb molecules interact with each other. The rate constants also decreased for K56C and A144C Mbs presumably because of the electrostatic repulsion during dimer formation, since they are relatively charged around the inserted cysteine.
人类肌红蛋白（Mb）有一个独特的半胱氨酸（Cys110），而其他哺乳动物的肌红蛋白则没有。为了研究半胱氨酸残基对肌红蛋白的影响，我们通过突变将半胱氨酸引入抹香鲸肌红蛋白表面的不同位点（K56C、V66C、K96C、K102C、A125C和A144C）。除了V66C的半胱氨酸被引入α-螺旋的中间位置外，其他半胱氨酸都被插入到α-螺旋的末端附近。在37℃的一氧化碳气氛下孵育时，观察到每个突变体高铁肌红蛋白（metMb）的血红素都发生了还原，这比在相同条件下野生型高铁肌红蛋白的还原速度要快得多。在氮气或氧气气氛下，血红素的还原没有明显发生。血红素还原的速率常数随着突变体肌红蛋白浓度的升高而增加，而当一氧化碳浓度从100% CO降至50% CO并含有50% O₂时，速率常数没有明显变化。不同一氧化碳浓度下速率常数的相似性表明，一氧化碳通过与血红素铁配位来稳定还原态的血红素。SDS-PAGE分析表明，突变体肌红蛋白二聚体是在一氧化碳气氛下孵育形成的，而在空气中则不会形成。通过添加2-巯基乙醇，这些二聚体又转化回肌红蛋白单体，这表明肌红蛋白二聚体是通过二硫键形成的。一般来说，随着血红素-半胱氨酸距离的增加，速率常数会降低，尽管V66C肌红蛋白的速率常数非常小。由于V66位于α-螺旋的中间位置，当两个不同的V66C肌红蛋白分子的半胱氨酸残基相互作用时，会发生空间位阻并阻止二聚体的形成。K56C和A144C肌红蛋白的速率常数也降低了，可能是因为在二聚体形成过程中的静电排斥作用，因为它们在插入的半胱氨酸周围带有相对电荷。
