Ivancich A, Barynin V V, Zimmermann J L
CEA, Centre d'Etudes de Saclay, DBCM, Section de Bioénergétique, Gif-sur-Yvette, France.
Biochemistry. 1995 May 23;34(20):6628-39. doi: 10.1021/bi00020a008.
The nature of possible protein ligands to the binuclear metal core in manganese catalase from Thermus thermophilus has been addressed by EPR and ESEEM (pulsed EPR) spectroscopies. The three-pulse ESEEM spectrum of the superoxidized Mn(III)Mn(IV) enzyme obtained at 3429 G shows a frequency pattern with peaks at 0.60, 1.45, 2.06, and 5.03 MHz that is assigned to the magnetic coupling in the exact cancellation regime of one 14N atom that coordinates the Mn dimer, with magnetic parameters e2Qq = 2.34 MHz, eta = 0.51, and Aiso = 2.45 MHz. When the enzyme is chemically modified by reductive methylation, dramatic effects are detected both in the CW-EPR spectrum and in the ESEEM data. Spectral simulations of the CW-EPR signal suggest that the alterations in the spectra are related to the properties of the hyperfine coupling tensors of the Mn ions and of the g tensor, which changes from axial symmetry (gparallel - gperpendicular = 0.018) in the untreated catalase to a nearly isotropic symmetry (gparallel - gperpendicular = 0.002) in the modified enzyme. The three-pulse ESEEM spectrum of the catalase is also completely altered after the reductive methylation, with a rather different frequency pattern at 1.57, 2.35, 3.88, and 6.00 MHz. These data are interpreted as indicating that the hyperfine interaction from the coupled 14N donor is profoundly modified by the methylation treatment, changing from Aiso = 2.45 MHz to a larger value. The spectra are compared with ESEEM data obtained on two polynuclear Mn systems with 14N donors: the Mn cluster of Photosystem II inhibited by 14NH4Cl, and the model compound Mn2(bipy)4(mu-O)23. It is found that the ESEEM data measured on the untreated Mn(III)Mn(IV) catalase resemble those on the Photosystem II manganese site, suggesting that the coupled 14N coordinates the Mn dimer in an analogous fashion. By analogy to the mode of binding of ammonia in Photosystem II proposed by Britt et al. [Britt, R. D., Zimmermann, J. L., Sauer, K., & Klein, M. P. (1989) J. Am. Chem. Soc. 111, 3522-3532], it is proposed that a 14N atom bridges the two Mn ions in Mn(III)Mn(IV) catalase. By contrast, comparison of the data obtained on the methylated enzyme with those on the model compound suggests that the 14N couplings are similar in both systems; this is indicative of a terminal 14N ligand in the modified catalase.(ABSTRACT TRUNCATED AT 400 WORDS)
嗜热栖热菌锰过氧化氢酶中双核金属核心可能的蛋白质配体的性质已通过电子顺磁共振（EPR）和电子自旋回波包络调制（ESEEM，脉冲EPR）光谱进行了研究。在3429 G下获得的超氧化态Mn(III)Mn(IV)酶的三脉冲ESEEM光谱显示出频率模式，其峰值分别位于0.60、1.45、2.06和5.03 MHz，这被归因于与Mn二聚体配位的一个14N原子在精确抵消状态下的磁耦合，其磁参数为e2Qq = 2.34 MHz，eta = 0.51，Aiso = 2.45 MHz。当该酶通过还原甲基化进行化学修饰时，在连续波EPR光谱和ESEEM数据中均检测到显著影响。连续波EPR信号的光谱模拟表明，光谱的变化与Mn离子的超精细耦合张量以及g张量的性质有关，g张量从未经处理的过氧化氢酶中的轴对称性（g平行 - g垂直 = 0.018）变为修饰后酶中的近各向同性对称性（g平行 - g垂直 = 0.002）。还原甲基化后，过氧化氢酶的三脉冲ESEEM光谱也完全改变，在1.57、2.35、3.88和6.00 MHz处呈现出相当不同的频率模式。这些数据被解释为表明耦合的14N供体的超精细相互作用因甲基化处理而被深刻改变，从Aiso = 2.45 MHz变为更大的值。将这些光谱与在两个具有14N供体的多核Mn系统上获得的ESEEM数据进行了比较：被14NH4Cl抑制的光系统II的Mn簇，以及模型化合物Mn2(bipy)4(μ - O)23。结果发现，在未经处理的Mn(III)Mn(IV)过氧化氢酶上测得的ESEEM数据与光系统II锰位点上的数据相似，这表明耦合的14N以类似方式与Mn二聚体配位。类比于Britt等人[Britt, R. D., Zimmermann, J. L., Sauer, K., & Klein, M. P. (1989) J. Am. Chem. Soc. 111, 3522 - 3532]提出的光系统II中氨的结合模式，推测在Mn(III)Mn(IV)过氧化氢酶中一个14N原子桥接两个Mn离子。相比之下，将甲基化酶上获得的数据与模型化合物上的数据进行比较表明，两个系统中的14N耦合相似；这表明修饰后的过氧化氢酶中有一个末端14N配体。（摘要截断于400字）
