Black S D
Department of Biochemistry, University of Texas Health Center at Tyler 75710-2003.
Biochem Biophys Res Commun. 1994 Aug 30;203(1):162-8. doi: 10.1006/bbrc.1994.2163.
Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium. The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain. In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome. This fragment bound the 2',5'-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, epsilon 453nm = 11,200 M-1cm-1). Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation. In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH. semiquinone radical. At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed. Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues). These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.
细胞色素P450 102是一种从巴比妥酸盐诱导的巨大芽孢杆菌中分离出来的具有催化自足性的单加氧酶。该酶在一条由1048个残基组成的单多肽链中含有黄素腺嘌呤二核苷酸（FAD）、黄素单核苷酸（FMN）和血红素，并且每个辅因子都被认为位于一个单独的结构域中。在本研究中，我们使用了彻底的内源性蛋白酶解来产生细胞色素的一个45 kDa片段。该片段与烟酰胺腺嘌呤二核苷酸磷酸（NADP(H)）的2',5'-二磷酸腺苷部分紧密结合，解离常数与天然酶中的大致相同，并且仅含有FAD（每个多肽0.93当量，ε453nm = 11,200 M-1cm-1）。连二亚硫酸钠对黄素的还原进行得相当缓慢，生成FADH2，但在空气再氧化时没有产生稳定的半醌物种。相反，NADPH能在有氧条件下迅速将这个FAD/NADP(H)结构域还原，产生FADH半醌自由基。当FAD/NADP(H)结构域与P450 102血红素结构域的摩尔比为75:1时，未观察到月桂酸羟化酶活性。气相序列分析表明存在两个主要序列，分别从苯丙氨酸646（403个残基，分子量45,033）和天冬氨酸652（397个残基）开始。这些数据与相关酶的晶体结构一致，并紧密界定了P450 102中FAD/NADP+结构域的边界。
