Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry
Observation of the iron-sulfur cluster in Escherichia coli biotin synthase by nanoflow electrospray mass spectrometry
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DOI：
10.1021/ac0102664
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发表时间：
2001-09-01
影响因子：
7.4
通讯作者：
Robinson, CV
中科院分区：
文献类型：
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作者：
Hernández, H;Hewitson, KS;Robinson, CV
采用纳米流电喷雾电离质谱仪对大肠杆菌生物素合成酶进行了分析。在蛋白质处于天然状态的溶液条件下，观察到单体、二聚体和四聚体物种的分布。这些物种的分布对离子强度的变化很敏感：在阳离子光谱中，在低离子强度(pH 7.0-8.5)下，生物素合成酶产生的二聚体不到10%。单体物种的质量与存在的[2Fe-2S]团簇相一致，与具有一个二硫键的非单体分子质量相差175.3 Da。尽管非共价二聚体的分子质量为77 kDa，但在正负离子光谱中仍有可能观察到含有一个铁硫簇合物的二聚体物种。此外，对脱辅基二聚体的一系列电荷状态的观察表明，铁-硫簇的结合不是维持二聚体所必需的。还观察到Cu2+与生物素合成酶的结合；在过量的螯合剂存在下，游离金属被去除，而铁-硫簇保持不变。在串联质谱学实验中获得了生物素合成酶中铁-硫簇配位的证据。分离出m/z为2416.9的团簇的单电荷态，碰撞诱导解离导致硫的顺序损失和Fe3+的保留。
Biotin synthase from Escherichia coli was analyzed by nanoflow electrospray ionization mass spectrometry. From solution conditions in which the protein is in its native state, a distribution of monomeric, dimeric, and tetrameric species was observed. The distribution of these species was sensitive to changes in ionic strength: in the positive ion spectrum, biotin synthase at low ionic strength (pH 7.0-8.5) yielded less than 10% dimer. The masses of the monomeric species were consistent with the presence of a [2Fe-2S] cluster with a mass difference of 175.3 Da from the apomonomer with one disulfide bond. Despite the molecular mass of the noncovalent dimer (77 kDa), it was possible to observe a dimeric species containing one iron-sulfur cluster in both positive and negative ion spectra. Additionally, observation of a series of charge states assigned to the apodimer indicated that binding of the iron-sulfur cluster was not required to maintain the dimer. Binding of Cu2+ to biotin synthase was also observed; in the presence of excess chelating agent, free metals were removed and the iron-sulfur cluster remained intact. Evidence for the coordination of the iron-sulfur cluster in biotin synthase was obtained in a tandem mass spectrometry experiment. A single charge state containing the cluster at m/z 2416.9 was isolated, and collision-induced dissociation resulted in sequential loss of sulfur and retention of Fe3+.