Mössbauer studies of the iron-sulfur cluster-free hydrogenase: the electronic state of the mononuclear Fe active site.
Mössbauer studies of the iron-sulfur cluster-free hydrogenase: the electronic state of the mononuclear Fe active site.
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DOI：
10.1021/ja051895o
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发表时间：
2005-07
影响因子：
15
通讯作者：
S. Shima;Erica J. Lyon;R. Thauer;Bernd Mienert;E. Bill
中科院分区：
文献类型：
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作者：
S. Shima;Erica J. Lyon;R. Thauer;Bernd Mienert;E. Bill
产甲烷古菌的铁硫簇自由氢化酶（Hmd）含有一个结构未知的含铁光敏辅基。该酶被CO和氰化物可逆地抑制，并且是EPR沉默的。我们在这里报告穆斯堡尔谱的（57）铁标记的酶和孤立的辅因子。在80 K下测得的全酶光谱显示出一个双峰，其异构体位移Δ = 0.06 mm·s（-）（1），四极分裂Δ E（Q）= 0.65 mm·s（-）（1）（pH 8.0）。信号强度对应于假定每摩尔活性位点1个Fe的酶浓度。在酶中加入CO或氰化物后，异构体位移分别降低到-0.03 mm·s（-）（1）和-0.00（1）mm·s（-）（1），四极分裂分别增加到1.38 mm·s（-）（1）和1.75 mm·s（-）（1）。假设HMD中仅存在一种类型的铁，则可以完美地模拟这三种光谱。低同分异构体位移是Fe处于低氧化态（0，+1，+2）的特征。当全酶和CO或氰化物抑制酶的光谱在4 K下在4和7 T的磁场中测量时，所获得的光谱可以模拟假设仅存在外部磁场，这排除了Hmd活性位点中的铁是Fe（I）、高自旋Fe（0）或高自旋Fe（II）。还报道了分离的HMD辅因子的穆斯堡尔谱。
The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing, light-sensitive cofactor of still unknown structure as prosthetic group. The enzyme is reversibly inhibited by CO and cyanide and is EPR silent. We report here on Mössbauer spectra of the (57)Fe-labeled enzyme and of the isolated cofactor. The spectrum of the holoenzyme measured at 80 K revealed a doublet peak with an isomer shift delta = 0.06 mm.s(-)(1) and a quadrupole splitting of DeltaE(Q) = 0.65 mm.s(-)(1) (at pH 8.0). The signal intensity corresponded to the enzyme concentration assuming 1 Fe per mol active site. Upon addition of CO or cyanide to the enzyme, the isomer shift decreased to -0.03 mm.s(-)(1) and -0.00(1) mm.s(-)(1), and the quadrupole splitting increased to 1.38 mm.s(-)(1) and 1.75 mm.s(-)(1), respectively. The three spectra could be perfectly simulated assuming the presence of only one type of iron in Hmd. The low isomer shift is characteristic for Fe in a low oxidation state (0, +1, +2). When the spectra of the holoenzyme and of the CO- or cyanide-inhibited enzyme were measured at 4 K in a magnetic field of 4 and 7 T, the spectra obtained could be simulated assuming the presence of only the external magnetic field, which excludes that the iron in the active site of Hmd is Fe(I), high-spin Fe(0), or high-spin Fe(II). Mössbauer spectra of the isolated Hmd cofactor are also reported.