Pochapsky Susan Sondej, Dang Marina, OuYang Bo, Simorellis Alana K, Pochapsky Thomas C
Department of Chemistry, Brandeis University, 415 South Street, MS 015, Waltham, Massachusetts 02454-9110, USA.
Biochemistry. 2009 May 26;48(20):4254-61. doi: 10.1021/bi900002k.
Local protein backbone dynamics of the camphor hydroxylase cytochrome P450(cam) (CYP101) depend upon the oxidation and ligation state of the heme iron. (1)H-(15)N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H-D exchange) and (15)N relaxation measurements, and these results are compared with previously published results obtained by H-D exchange mass spectrometry. In general, the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B' helix and beta3 and beta5 sheets) and binding of putidaredoxin (C and L helices), the iron-sulfur protein that acts as the effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and the relevance of the observed effects to the enzyme mechanism is discussed.
樟脑羟化酶细胞色素P450（cam）（CYP101）的局部蛋白质主链动力学取决于血红素铁的氧化和连接状态。利用（1）H-（15）N相关核磁共振实验，通过氢-氘交换动力学（H-D交换）和（15）N弛豫测量，比较了这种414个残基的金属酶氧化态和还原态的主链动力学，并将这些结果与先前通过H-D交换质谱获得的结果进行了比较。一般来说，在这些实验可及的所有时间尺度上，还原态酶的二级结构特征运动幅度低于氧化态酶，并且这些差异在参与底物进入活性位点的酶区域（B'螺旋以及β3和β5折叠）和恶臭假单胞菌铁氧还蛋白（C和L螺旋）的结合区域更为明显，恶臭假单胞菌铁氧还蛋白是一种铁硫蛋白，在体内作为CYP101的效应物和还原剂。这些结果根据血红素氧化态变化的局部结构效应进行了解释，并讨论了观察到的效应与酶机制的相关性。
