METHANE MONOOXYGENASE STRUCTURE AND MECHANISM

我们建议研究活性中心的结构和 甲烷单加氧酶催化作用的分子机理 (MMO)从II型甲烷氧化菌Methylosinus纯化 发孢霉（Trichosporium OB3b）OB3b）。 这种酶催化第一种 甲烷的细菌氧化步骤（产生甲醇） 并将偶然催化许多其它的羟基化反应， 饱和和不饱和烃。 这3个机制 酶系统的组成是未知的。 然而，研究 这种酶以及从其他2个来源分离的MMO 表明实际上催化 羟基化不含已知存在于 其他单加氧酶。 因此，MMO可能会使用一个新的 催化机制。 我们已经提纯了所有的成分 关于M. T OB3b MMO。 该系统具有显著的优势 超过2个其他纯化MMO系统，包括更大的稳定性， 更高的产量和比活性增加15倍。 这些 性质将允许克量的纯化 酶，使生物物理技术，包括光学，EPR， 穆斯堡尔谱和EXAFS谱可用于确定 羟化酶活性部位的结构。 光谱 小配体络合物，基于机理的抑制剂，同位素 标记的底物和抑制剂，以及瞬时动力学， 作为研究分子机制的方法。 这项工作应该产生一个新的类型的基本理解 氧活化化学，铁在这一新的作用， 化学，也许是对催化剂设计的新见解， 大量碳氢化合物的氧化。

We propose to investigate the active site structure and the molecular mechanism of catalysis by Methane Monooxygenase (MMO) purified from the Type II methanotroph, Methylosinus trichosporium OB3b (M.t. OB3b). This enzyme catalyzes the first step in the bacterial oxidation of methane (producing methanol) and will adventitiously catalyze hydroxylation of many other saturated and unsaturated hydrocarbons. The mechanism of this 3 component enzyme system is unknown. However, studies with this enzyme as well as MMOs isolated from 2 other sources suggest that the component which actually catalyzes the hydroxylation contains none of the cofactors known to exist in other monooxygenases. Thus MMO probably uses a new mechanism for catalysis. We have purified all of the components of M.t. OB3b MMO. The system offers significant advantages over the 2 other purified MMO systems including greater stability, greater yield and a 15-fold increase in specific activity. These properties will allow the purification of gram quantities of enzyme so that biophysical techniques including optical, EPR, Mossbauer and EXAFS spectroscopies can be applied to determine the structure of the hydroxylase active site. Spectroscopy of small ligand complexes, mechanism based inhibitors, isotopically labeled substrates and inhibitors, and transient kinetics are proposed as methods to investigate the molecular mechanism. This work should yield a fundamental understanding of a new type of oxygen activation chemistry, a new role for Fe in this chemistry and perhaps new insight into the design of catalysts for oxidation of abundant hydrocarbons.