Effect of iron-sulfur cluster environment in modulating the thermodynamic properties and biological function of ferredoxin from Pyrococcus furiosus
Effect of iron-sulfur cluster environment in modulating the thermodynamic properties and biological function of ferredoxin from Pyrococcus furiosus
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DOI：
10.1021/bi972864b
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发表时间：
1998-05-19
期刊：
影响因子：
2.9
通讯作者：
Adams, MWW
中科院分区：
文献类型：
--
作者：
Brereton, PS;Verhagen, MFJM;Adams, MWW
超嗜热古菌Pyrococcus furiosus的铁氧还蛋白（7.5 kDa）含有一个由三个Cys和一个Asp残基而不是预期的四个Cys配位的[4Fe-4S]（1+，2+）簇。使用一系列突变体D14 X（其中X = C、S、H、N、V和Y）通过在大肠杆菌中异源基因表达制备，研究了此Asp残基的作用。而野生型和D14 S和D14 C突变体的重组形式含有一个[4Fe-4S]（1+，2+）簇，D14 V，D14 H，D14 Y，和D14 N蛋白含有一个[3Fe-4S]（0，+）中心，通过可见光谱和电化学测定。氧化还原电位与野生型4Fe-铁氧还蛋白相比，D14 C和D14 S突变体的铁氧还蛋白的氧化还原电位（pH 7.0，23 ℃）分别降低了58和133 mV（E-m-368 mV），而3Fe-蛋白突变体（包括D14S的3Fe-形式，通过化学氧化产生）比野生型3Fe-形式（通过化学氧化获得，E-m-203 mV）的正性高15 - 118 mV。除D14S突变体外，所有3Fe形式的还原电位均显示pH响应范围为3.0 - 10.0，pK为3.3 - 4.7，这归因于簇质子化。D14 H突变体和野生型3Fe-蛋白质显示出额外的pK（均为5.9），假定其由氨基酸侧链的质子化引起。与4Fe-蛋白质，有野生型或D14 C形式的电位没有显着的变化，而D14 S突变体（pK 4.75）的pH响应归因于丝氨酸的质子化。虽然铁氧还蛋白变体表现出一定范围的热稳定性（在80 ℃，pH 2.5下测量），但它们中没有一个在其还原电位中显示出任何温度依赖性转变（0 - 80 ℃），并且在计算的Δ S度值与残基14的最大吸光度、还原电位或疏水性之间没有相关性。相反，Δ H度值与还原电位之间存在线性相关。在80 ℃下使用铁氧还蛋白作为丙酮酸氧化还原酶（POR）的电子受体或作为铁氧还蛋白：NADP氧化还原酶（FNOR，两者均来自P.furiosus）的电子供体进行动力学分析.数据表明，铁氧还蛋白的还原电位，而不是簇类型或在位置14处的残基的性质，似乎是决定两个系统中的电子转移效率的主要因素。然而，与所有变体相比，WT Fd的还原电位使其成为最适合从FOR接受电子并将其捐赠给FNOR的蛋白质。
The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single [4Fe-4S](1+,2+) cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys. The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli. While the recombinant form of the wild-type and the D14S and D14C mutants contained a [4Fe-4S](1+,2+) cluster, the D14V, D14H, D14Y, and D14N proteins contained a [3Fe-4S](0,+) center, as determined by visible spectroscopy and electrochemistry. The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (E-m -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, E-m -203 mV). The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation. The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain. With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate. While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated Delta S degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14. In contrast, there was a linear correlation between the Delta H degrees' value and reduction potential. Kinetic analyses were carried out at 80 degrees"C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P. furiosus). The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems. However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from FOR and donate them to FNOR.