Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.
Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.
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DOI：
10.1021/bi300664j
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发表时间：
2012-10-16
期刊：
影响因子：
2.9
通讯作者：
Johnson MK
中科院分区：
文献类型：
--
作者：
Mapolelo DT;Zhang B;Naik SG;Huynh BH;Johnson MK
为了评估铁结合形式在nif特异性Fe- s簇生物发生中的作用，研究了固氮菌vinelandii NifIscA结合铁的能力。NifIscA显示每个同型二聚体结合一个Fe（III）或一个Fe(II)，并且使用紫外可见吸收，CD和VTMCD， EPR， Mössbauer和共振拉曼光谱表征了Fe(III)-和Fe（II）结合形式的光谱和氧化还原性质。结果表明，菱形中间自旋（S = 3/2） Fe（III）中心（E/D = 0.33, D = 3.5±1.5cm−1）与2个或3个半胱氨酸配体最可能是5配位；菱形高自旋（S = 2） Fe（II）中心（E/D = 0.28, D = 7.6 cm−1）具有与3个半胱氨酸配体和1或2个含氧配体相似的还原红霉素或红霉素变体的性质。铁结合的NifIscA在pH 7.8（相对于NHE）下，在Fe(III)/Fe（II）形态之间进行可逆的氧化还原循环，中点电位为+36±15 mV。l -半胱氨酸可以有效地介导游离铁（II）从铁(II)-和铁（III）结合形式的NifIscA释放。Fe（III）结合的NifIscA也被证明是NifU n端结构域上nifs介导的[2Fe-2S]簇组装的有效铁源，但该反应是通过半胱氨酸介导的游离Fe（II）释放而不是直接铁转移发生的。根据这些结果，讨论了a型蛋白在有氧生长条件下储存铁的作用，以及作为u型支架蛋白簇组装或生物[4Fe-4S]中心成熟的铁供体。
The ability of Azotobacter vinelandii NifIscA to bind Fe has been investigated to assess the role of Fe-bound forms in NIF-specific Fe-S cluster biogenesis. NifIscA is shown to bind one Fe(III) or one Fe(II) per homodimer and the spectroscopic and redox properties of both the Fe(III)- and Fe(II)-bound forms have been characterized using the UV-visible absorption, CD and VTMCD, EPR, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic intermediate-spin (S = 3/2) Fe(III) center (E/D = 0.33, D = 3.5 ± 1.5cm−1) that is most likely 5-coordinate with two or three cysteinate ligands and a rhombic high spin (S = 2) Fe(II) center (E/D = 0.28, D = 7.6 cm−1) with properties similar to reduced rubredoxins or rubredoxin variants with three cysteinate and one or two oxygenic ligands. Iron-bound NifIscA undergoes reversible redox cycling between the Fe(III)/Fe(II) forms with a midpoint potential of +36 ±15 mV at pH 7.8 (versus NHE). L-cysteine is effective in mediating release of free Fe(II) from both the Fe(II)- and Fe(III)-bound forms of NifIscA. Fe(III)-bound NifIscA was also shown to a competent iron source for in vitro NifS-mediated [2Fe-2S] cluster assembly on the N-terminal domain of NifU, but the reaction occurs via cysteine-mediated release of free Fe(II) rather than direct iron transfer. The proposed roles of A-type proteins in storing Fe under aerobic growth conditions and serving as iron donors for cluster assembly on U-type scaffold proteins or maturation of biological [4Fe-4S] centers are discussed in light of these results.
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