MOSSBAUER, EPR, AND ENDOR STUDIES OF THE HYDROXYLASE AND REDUCTASE COMPONENTS OF METHANE MONOOXYGENASE FROM METHYLOSINUS-TRICHOSPORIUM OB3B
MOSSBAUER, EPR, AND ENDOR STUDIES OF THE HYDROXYLASE AND REDUCTASE COMPONENTS OF METHANE MONOOXYGENASE FROM METHYLOSINUS-TRICHOSPORIUM OB3B
复制标题
DOI：
10.1021/ja00062a039
复制
发表时间：
1993-05-05
影响因子：
15
通讯作者：
MUNCK, E
中科院分区：
文献类型：
--
作者：
FOX, BG;HENDRICH, MP;MUNCK, E
从发孢甲基弯菌（Methylosinus trichosporium）OB 3 B中分离得到的可溶性甲烷单加氧酶（MMO）由三种组分组成：羟化酶、还原酶和组分B。用穆斯堡尔谱、ENDOR谱和EPR谱研究了羟化酶活性中心的二铁团簇。氧化团簇的穆斯堡尔谱表明，两个高自旋铁是反铁磁耦合的，这与我们的初步研究雅阁（Fox等，J. Biol. Chem. 1988，263，10553-10556）。穆斯堡尔谱研究还揭示了在pH 9时存在两种簇构象。交换耦合团簇（Fe ~（3+）. Fe ~（3+））的激发态S = 2多重态在g = 8附近产生整数自旋EPR信号;这是首次对来自任何系统的这种信号进行定量研究。对g = 8信号的温度依赖性的分析得出交换耦合常数J = 15 +/- 5 cm-1（H（ex）= JS1.S2）。该值比报道的血红蛋白和大肠杆菌核糖核苷酸还原酶的氧桥簇（H（ex）= JS1.S2，J = 270和220 cm-1）小1个数量级以上，表明羟化酶簇的桥连配体不是未取代的氧原子。穆斯堡尔谱的羟化酶在应用领域的高达8 T揭示了一个顺磁混合物的低洼激发态到地面单。假设自旋期望值随磁场线性增加，谱的形状和强度都得到了很好的描述。然而，这种效应的起源是不能完全解释的标准自旋哈密顿量的框架下，包括零场分裂和反对称交换。未络合的混合价态（Fe ~（3+）. Fe ~（2+））羟化酶的EPR研究表明，该酶由两种稍有不同的簇合物组成，其比例约为4：1。混合价羟化酶亚铁位点的零场分裂（zero-field splitting，EFF）对与产物或抑制剂的络合敏感，而组分B的络合干扰交换耦合。抑制剂二甲基亚砜的结合导致ZFS参数的最小分布，因此在这里使用三种光谱技术中的每一种进行相关研究。数据进行了分析与自旋哈密顿量，包括交换耦合（J = 60 cm-1）和混合的多重态的零场分裂。分析表明，铁的网站的轨道基态具有主要的d（xy）对称性，该轨道的z轴点沿着z方向的集群g-张量。穆斯堡尔谱和Fe-57-ENDOR谱表明铁原子的A张量是各向异性的，Fe-57-ENDOR信号是首次报道的二铁氧簇。在强磁场（高达6.0 T）下记录的未络合的还原（Fe ~（2+）. Fe ~（2+））羟化酶簇的穆斯堡尔谱分析明确地表明两个铁位点是不等价的。氧化簇的光谱也是最好的拟合假设集群的铁驻留在不等价的环境。考虑到正在进行的结构研究所揭示的羟化酶的整体双重对称性，目前的研究结果表明，羟化酶包含两个，可能是相同的，活性位点二铁簇，其个别的铁原子结构不同。还报道和分析了MMO还原酶组分[2Fe-2S]2+，1+簇合物的穆斯堡尔谱和EPR谱。
Soluble methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b consists of three components: hydroxylase, reductase, and component B. The active-site diiron cluster of the hydroxylase has been studied with Mossbauer, ENDOR, and EPR spectroscopies. Mossbauer spectra of the oxidized cluster show that the two high-spin irons are antiferromagnetically coupled in accord with our preliminary study (Fox et al. J. Biol. Chem. 1988, 263, 10553-10556). Mossbauer studies also reveal the presence of two cluster conformations at pH 9. The excited-state S = 2 multiplet of the exchange-coupled cluster (Fe3+.Fe3+) gives rise to an integer-spin EPR signal near g = 8; this is the first quantitative study of such a signal from any system. Analysis of the temperature dependence of the g = 8 signal yields J = 15 +/- 5 cm-1 for the exchange-coupling constant (H(ex) = JS1.S2). This value is more than 1 order of magnitude smaller than those reported for the oxo-bridged clusters of hemerythrin and Escherichia coli ribonucleotide reductase (H(ex) = JS1.S2, J = 270 and 220 cm-1, respectively), suggesting that the bridging ligand of the hydroxylase cluster is not an unsubstituted oxygen atom. Mossbauer spectra of the hydroxylase in applied fields of up to 8 T reveal a paramagnetic admixture of a low-lying excited state into the ground singlet. Both the spectral shape and intensity are well represented by assuming that the spin expectation values for the cluster sites increase linearly with magnetic field. However, the origin of this effect is not fully explicable in the framework of the standard spin Hamiltonian including zero-field splittings and antisymmetric exchange. EPR studies of the uncomplexed mixed valence (Fe3+.Fe2+) hydroxylase show that it is composed of two slightly different cluster forms in an approximate 4:1 ratio. The zero-field splitting (ZFS) of the ferrous site of the mixed valence hydroxylase is sensitive to complexation with products or inhibitors, while complexation by the component B perturbs the exchange coupling. The binding of the inhibitor dimethyl sulfoxide results in the smallest distribution of ZFS parameters and thus is investigated here in a correlated study using each of the three spectroscopic techniques. The data were analyzed with a spin Hamiltonian that includes exchange coupling (J = 60 cm-1) and mixing of multiplets by zero-field splittings. The analysis shows that the orbital ground state of the ferrous site has predominantly d(xy) symmetry; the z-axis of this orbital points along the z-direction of the cluster g-tensor. Mossbauer and Fe-57-ENDOR spectra indicate that the A-tensor of the ferric site is anisotropic; the Fe-57-ENDOR signals are the first reported for diiron-oxo clusters. Analysis of the Mossbauer spectra of the uncomplexed, reduced (Fe2+.Fe2+) hydroxylase cluster recorded in strong applied fields (up to 6.0 T) unambiguously shows that the two iron sites are inequivalent. Spectra of the oxidized cluster are also best fit by assuming that the irons of the cluster reside in inequivalent environments. Considered in light of the overall two-fold symmetry of hydroxylase revealed by ongoing structural studies, the present findings show that the hydroxylase contains two, probably identical, active-site diiron clusters whose individual iron atoms are structurally distinct. Mossbauer and EPR spectra of the [2Fe-2S]2+,1+ cluster of the MMO reductase component are also reported and analyzed.