Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere
Heme reduction by intramolecular electron transfer in cysteine mutant myoglobin under carbon monoxide atmosphere
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DOI：
10.1021/bi0507581
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发表时间：
2005-08-02
期刊：
影响因子：
2.9
通讯作者：
Funasaki, N
中科院分区：
文献类型：
--
作者：
Hirota, S;Azuma, K;Funasaki, N
人类肌红蛋白（Mb）具有独特的半胱氨酸（Cys110），而其他哺乳动物的Mb则没有。为了研究半胱氨酸残基对Mb的影响，我们通过突变将半胱氨酸引入抹香鲸Mb表面的不同位点（K56C、V66C、K96C、K102C、A125C和A144C）。半胱氨酸。在a-螺旋的末端插入半胱氨酸，除了V66C，在c-螺旋的中间插入半胱氨酸。在37℃的一氧化碳气氛下，每个突变体memb的血红素还原速度都比野生型memb的还原速度快得多。在氮气或氧气环境下，血红素还原不明显。突变体Mb浓度越高，血红素还原速率常数越高，而当CO浓度从100% CO降低到50% CO， 50% O-2时，血红素还原速率常数变化不显著。不同CO浓度下速率常数的相似性表明，CO通过与血红素铁的配位来稳定还原血红素。SDS-PAGE分析表明，突变体Mb二聚体是在CO气氛下形成的，而不是在空气中形成的。这些二聚体通过添加2-巯基乙醇转化回Mb单体，这表明通过二硫键形成Mb二聚体。随着血红素-半胱氨酸距离的增加，速率常数普遍降低，但V66C Mb的速率常数很小。由于V66位于α -螺旋的中间，当两种不同V66C Mb分子的半胱氨酸残基相互作用时，会发生位阻，阻止二聚体的形成。K56C和A144C Mbs的速率常数也降低了，这可能是由于二聚体形成过程中的静电斥力，因为它们在插入的半胱氨酸周围相对带电。
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 a-helices, except for V66C, where the cysteine was introduced in the middle of an cc-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.