Fox B G, Surerus K K, Münck E, Lipscomb J D
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
J Biol Chem. 1988 Aug 5;263(22):10553-6.
Mössbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The Mössbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the Mössbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.
报道了对从甲基孢囊菌OB3b中分离出的甲烷单加氧酶高活性羟化酶组分进行的穆斯堡尔谱和电子顺磁共振（EPR）研究。氧化态（刚分离出时）羟化酶的穆斯堡尔谱表明，铁存在于一个抗磁性簇中，该簇包含偶数个Fe3+位点。这些参数与类似于蚯蚓血红蛋白和紫色酸性磷酸酶的反铁磁耦合双核簇一致。羟化酶部分还原后，观察到一个S = 1/2的EPR谱，其g值分别为1.94、1.86和1.75（gav = 1.85）。这种谱是混合价态Fe(II).Fe(III)状态下氧桥联铁二聚体的特征。进一步还原导致在g = 15处出现一个新的EPR共振。与μ-氧桥联双核铁的无机模型化合物比较表明，g = 15的信号是蛋白质中簇的双还原态的特征。在这种状态下，穆斯堡尔谱呈现出高自旋Fe2+典型的两个四极双峰，与簇的Fe(II).Fe(II)形式一致。羟化酶铁中心在三种氧化态下的光谱特征都与报道的μ-氧（或μ-羟基）桥联双核铁簇相似。由于已知的单加氧酶都不包含这样的簇，因此提出了一种新的加氧酶机制。三种不同的制备方法得到的羟化酶比活性范围跨度为9倍，但观察到相同的簇浓度和光谱特征。因此，除了这里测量的参数外，其他参数对活性有重大影响。
