Leech Helen K, Raux Evelyne, McLean Kirsty J, Munro Andrew W, Robinson Nigel J, Borrelly Gilles P M, Malten Marco, Jahn Dieter, Rigby Stephen E J, Heathcote Peter, Warren Martin J
School of Biological Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
J Biol Chem. 2003 Oct 24;278(43):41900-7. doi: 10.1074/jbc.M306112200. Epub 2003 Aug 12.
CbiX is a cobaltochelatase required for the biosynthesis of vitamin B12 and is found in Archaea as a short form (CbiXS containing 120-145 amino acids) and in some bacteria as a longer version (CbiXL containing 300-350 amino acids). Purification of either recombinant Bacillus megaterium or Synechocystis CbiXL in Escherichia coli, which is facilitated by the presence of a naturally occurring histidine-rich region of the protein, results in the isolation of a dark brown protein solution. The UV/visible spectrum of the protein is consistent with the presence of a redox group, and the lack of definition within the spectrum is suggestive of a 4Fe-4S center. The presence of an iron-sulfur center was confirmed by EPR analysis of the proteins, which produces a pseudoaxial spectrum with g values at 2.04, 1.94, and 1.90. The EPR spectrum was absent at 70 K, an observation that is diagnostic of a 4Fe-4S center. Redox potentiometry coupled with optical spectroscopy allowed the midpoint potential of the redox center to be determined for the CbiXL from both B. megaterium and Synechocystis. Sequence analysis of CbiXL proteins reveals only two conserved cysteine residues within the CbiXL proteins, which are part of an MXCXXC motif. Mutagenesis of the two cysteines leads to loss of both the EPR spectrum and UV/visible spectral features of the Fe-S center in the protein, clearly indicating that these residues are involved in ligating the cofactor to the apoprotein possibly in a butterfly arrangement. The potential physiological role of the iron-sulfur center is discussed.
CbiX是维生素B12生物合成所需的钴螯合酶，在古菌中以短形式（含有120 - 145个氨基酸的CbiXS）存在，在一些细菌中以较长形式（含有300 - 350个氨基酸的CbiXL）存在。在大肠杆菌中纯化重组巨大芽孢杆菌或集胞藻的CbiXL，该蛋白天然存在富含组氨酸的区域有助于纯化，最终得到深棕色蛋白质溶液。该蛋白的紫外/可见光谱与氧化还原基团的存在一致，光谱中缺乏明确特征表明存在一个4Fe - 4S中心。通过对这些蛋白质的电子顺磁共振（EPR）分析证实了铁硫中心的存在，其产生一个伪轴向光谱，g值分别为2.04、1.94和1.90。在70 K时EPR光谱消失，这一观察结果可诊断为4Fe - 4S中心。氧化还原电位测定与光谱学相结合，使得能够确定来自巨大芽孢杆菌和集胞藻的CbiXL氧化还原中心的中点电位。CbiXL蛋白的序列分析显示，CbiXL蛋白中仅存在两个保守的半胱氨酸残基，它们是MXCXXC基序的一部分。对这两个半胱氨酸进行诱变导致该蛋白中铁硫中心的EPR光谱和紫外/可见光谱特征均丧失，这清楚地表明这些残基可能以蝶形排列参与将辅因子连接到脱辅基蛋白上。文中还讨论了铁硫中心潜在的生理作用。
