Cadieux Elisabeth, Vrajmasu Vladislav, Achim Catalina, Powlowski Justin, Münck Eckard
Department of Chemistry and Biochemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Quebec, Canada, H3G 1M8.
Biochemistry. 2002 Aug 27;41(34):10680-91. doi: 10.1021/bi025901u.
Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM:1 DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. Mössbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form I, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H(2)O(2) generation. In addition, Mössbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.
假单胞菌属CF600菌株的苯酚羟化酶由三个组分组成：DmpP是一种含FAD和[2Fe-2S]的还原酶；DmpM是一种无辅因子的激活蛋白；DmpLNO是加氧酶。单周转实验表明DmpLNO含有活性位点，但高效周转需要DmpM：稳态周转速率在1.5 DmpM:1 DmpLNO时达到最大值。化学交联实验表明DmpM与DmpLNO加氧酶复合物的大亚基相互作用。穆斯堡尔研究表明，加氧酶的活性位点可容纳两种类型的双铁簇，每种簇类型有两个等效位点。簇形式I通常占总铁的85%左右，ΔE(Q)=1.73 mm/s，δ=0.54 mm/s，而簇II的ΔE(Q)=0.79 mm/s，δ=0.48 mm/s。在强外加磁场中的研究表明，簇I的两个铁位点由一个氧桥连接，而簇II中的位点似乎由羟基桥连接。用连二亚硫酸盐还原样品可得到簇的二价铁形式。还原样品的空气氧化导致簇II部分增加，同时催化活性相应降低。还原的加氧酶样品在X波段呈现一个整数自旋EPR信号，在平行模式下，中心位于g = 16.6。定量分析表明，19%的簇对EPR信号有贡献，表明簇II是EPR活性物种。用二硫苏糖醇（DTT）孵育使加氧酶失活，其机制显然涉及H(2)O(2)的产生。此外，对DTT失活酶的穆斯堡尔研究表明，所有的铁离子都属于一个抗磁性的二价铁簇，其参数表明DTT与该簇配位。
