MOSSBAUER AND INTEGER-SPIN EPR OF THE OXIDIZED P-CLUSTERS OF NITROGENASE - POX IS A NON-KRAMERS SYSTEM WITH A NEARLY DEGENERATE GROUND DOUBLET
MOSSBAUER AND INTEGER-SPIN EPR OF THE OXIDIZED P-CLUSTERS OF NITROGENASE - POX IS A NON-KRAMERS SYSTEM WITH A NEARLY DEGENERATE GROUND DOUBLET
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DOI：
10.1021/ja00048a034
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发表时间：
1992-10-21
影响因子：
15
通讯作者：
MUNCK, E
中科院分区：
文献类型：
--
作者：
SURERUS, KK;HENDRICH, MP;MUNCK, E
固氮酶的钼铁蛋白含有2个Mo原子和约28-30个Fe原子。约有16个Fe原子属于P-团簇，这是一种结构未知的新型铁硫团簇。穆斯堡尔研究已经证实，P-团簇在半还原态P(N)中是抗磁的。蛋白质用硫代等氧化还原染料氧化后，得到P(OX)态。前人的研究表明，即使在没有外加磁场的情况下，P(OX)的低温(小于或等于4.2K)穆斯堡尔谱也表现出磁性超精细图案。这种行为通常是Kramers系统的特征，即具有奇数个电子的系统的特征。光谱具有Kramers二重态的典型特征，具有极各向异性的g值(g1>；>；g2，g3)。最近我们实验室对整数自旋系统的穆斯堡尔和EPR研究表明，P(OX)可能是一个非常不寻常的非Kramers系统。本文报道了棕色固氮菌(Av1)、巴氏梭菌(Cp1)、肺炎克雷伯氏菌(Kp1)和自养黄杆菌(Xa1)的蛋白质的穆斯堡尔谱和电子顺磁共振研究，证明P(Ox)的电子基对不是Kramers对，而是偶数电子体系的近简并对对。Cp1、Av1和Kp1具有小于或等于10(-3)cm-1的增量，零场穆斯堡尔谱中观察到的磁谱是两个电子能级通过Fe-57超精细相互作用(A(Z)几乎等于10(-3)cm-1)混合的结果。对于Av1和Kp1，我们观察到在g(Ef)=11.9时，在10-15 cm-1处的两个激发态之间的整数自旋EPR跃迁。在g(Jeff)=15.6时，XA1的基二重态表现出整数自旋共振。对XA1穆斯堡尔谱的分析得出了几乎等于-0.010厘米-1的增量。使用这个Delta的值，EPR谱的定量产生了P(Ox)的每个MoFe蛋白大约两个自旋。观测到的g值表明，XA1的P(OX)具有S=3或S=4。然而，Kp1和Av1的基态和激发态Delta值表明，电子基流形可能不是由具有一定自旋S的孤立多重态组成的。识别P(OX)为非Kramers态意味着在P(N)--Gt；P(Ox)的转变中每个P-团簇中移去了两个电子。由于每个MoFe蛋白约有4个电子被移除，由于涉及约16个Fe原子，因此固氮酶的Alphabeta二聚体包含两个相同的P-簇，每个簇具有约8个Fe位置。对于认为P星系团由两个桥接立方体组成的模型，穆斯堡尔数据进行了审查。
The molybdenum-iron protein of nitrogenase contains 2 Mo atoms and ca. 28-30 Fe atoms. Approximately 16 Fe atoms belong to the P-clusters, a novel type of iron-sulfur cluster of unknown structure. Mossbauer studies have established that P-clusters are diamagnetic in the semireduced state, P(N). Upon oxidation of the protein with redox dyes such as thionin the state P(OX) is attained. Previous studies have revealed that the low-temperature (less-than-or-equal-to 4.2 K) Mossbauer spectra of P(OX) exhibit magnetic hyperfine patterns even in the absence of external magnetic fields. Such behavior is generally characteristic of a Kramers system, i.e., of a system with an odd number of electrons. The spectra had features typical of those observed for a Kramers doublet with extremely anisotropic g-values (g1 >> g2, g3). Recent Mossbauer and EPR studies of integer spin systems in our laboratory have suggested the possibility that P(OX) may be a very unusual non-Kramers system. Here we report Mossbauer and EPR studies of the proteins from Azotobacter vinelandii (Av1), Clostridium pasteurianum (Cp1), Klebsiella pneumoniae (Kp1), and Xanthobacter autotrophicus (Xa1) which prove that the electronic ground doublet of P(OX) is not a Kramers doublet but rather a nearly degenerate doublet (splitting DELTA) of a system with an even number of electrons. Cp1, Av1, and Kp1 have DELTA less-than-or-equal-to 10(-3) cm-1, and the magnetic patterns observed in the zero-field Mossbauer spectra result from mixing of the two electronic levels by Fe-57 hyperfine interactions (\A(z)\ almost-equal-to 10(-3) cm-1). For Av1 and Kp1 we have observed integer-spin EPR transitions, at g(eff) = 11.9, between two excited-state spin levels at 10-15 cm-1. The ground doublet of Xa1 exhibits an integer-spin resonance at g(eff) = 15.6. Analysis of the Xa1 Mossbauer spectra yields DELTA almost-equal-to 0.010 cm-1. Using this value of DELTA, quantitation of the EPR spectra yielded ca. two spins per MoFe protein for P(OX). The observed g-values suggested that P(OX) of Xa1 has S = 3 or S = 4. However, the ground- and excited-state DELTA-values of Kp1 and Av1 indicate that the electronic ground manifold may not consist of an isolated multiplet with definite spin S. Recognition of P(OX) as a non-Kramers state implies that two electrons are removed from each P-cluster in the transformation P(N) --> P(OX). Since ca. four electrons per MoFe protein are removed and since ca. 16 Fe atoms are involved, it follows that the alphabeta dimer of nitrogenase contains two identical P-clusters and that each cluster has ca. 8 Fe sites. The Mossbauer data are reviewed for a model that considers the P-clusters to consist of two bridged cubanes.