Wang Sha, Liu Wei-Feng, He Yong-Zhi, Zhang Ao, Huang Li, Dong Zhi-Yang, Yan Yong-Bin
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100081, China.
Biochim Biophys Acta. 2008 Mar;1784(3):445-54. doi: 10.1016/j.bbapap.2007.12.001. Epub 2007 Dec 14.
Superoxide dismutases (SODs), which are the first line of cellular defense against the toxic effects of reactive oxygen species, are metalloenzymes that catalyze the disproportionation of superoxide radicals to produce oxygen and hydrogen peroxide. Although much effort has been devoted to the folding mechanisms of Cu/Zn-SODs, little is known about the folding of Fe-SODs. In this research, the equilibrium unfolding and refolding of TcSOD, a tetrameric hyperthermostable Fe-SOD, were investigated by circular dichroism, intrinsic fluorescence, ANS fluorescence, size-exclusion chromatography and cross-linking experiments. The results herein suggested that the guanidine hydrochloride-induced unfolding of TcSOD involved a stable monomeric intermediate and a possible tetrameric intermediate. The Gibbs free energy of TcSOD dissociation was about 3-fold larger than that of the monomeric intermediate unfolding, which suggested that the quaternary structure plays a crucial role in TcSOD stability. A comparison of the thermodynamic parameters between TcSOD and other SODs also suggested that the stability of quaternary structure might be responsible for the hyperthemostability of TcSOD.
超氧化物歧化酶（SODs）是细胞抵御活性氧毒性作用的第一道防线，是催化超氧阴离子自由基歧化生成氧气和过氧化氢的金属酶。尽管人们对铜/锌超氧化物歧化酶的折叠机制投入了大量精力，但对铁超氧化物歧化酶的折叠却知之甚少。在本研究中，通过圆二色性、内源荧光、ANS荧光、尺寸排阻色谱和交联实验研究了四聚体超嗜热铁超氧化物歧化酶TcSOD的平衡去折叠和重折叠过程。本文结果表明，盐酸胍诱导的TcSOD去折叠涉及一个稳定的单体中间体和一个可能的四聚体中间体。TcSOD解离的吉布斯自由能比单体中间体去折叠的吉布斯自由能大约3倍，这表明四级结构在TcSOD稳定性中起关键作用。TcSOD与其他超氧化物歧化酶热力学参数的比较也表明，四级结构的稳定性可能是TcSOD超嗜热性的原因。
