Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.
Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.
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通过对咪唑与荚膜红杆菌细胞色素 c2 铰链突变体结合的研究揭示了第六配体甲硫氨酸区域的蛋白质动力学。
DOI：
10.1021/bi0362370
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Cusanovich,MA
中科院分区：
文献类型：
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作者：
Dumortier,C;Fitch,J;VanPetegem,F;Vermeulen,W;Meyer,TE;VanBeeumen,JJ;Cusanovich,MA
迄今为止所研究的所有I类c型细胞色素在氧化状态下都经历了一个动态过程，导致铁-甲硫氨酸-硫键的短暂断裂和足够的运动以允许外源配体的结合（本研究中使用咪唑）。以荚膜红杆菌细胞色素c2为例，位于两个相对刚性螺旋区之间的第6血红素配体Met96和多达14个侧翼残基（位置88 - 100，称为铰链区）可能参与结构变化，导致能够结合配体的瞬时高自旋物种。我们检测了荚膜红杆菌细胞色素c2铰链区9个位置的14个突变，并确定了G95E突变体的结构。铰链区域的N端和c端附近的突变不影响运动动力学，但允许我们进一步定义铰链从血红素移动到氨基酸序列中93−100区域的部分。位置93和95的突变可以改变铰链运动的速率常数（高达20倍），可能是由于改变了天然细胞色素的结构，使其倾向于更开放的构象。其中一个突变体G95E的结构表明，铰链区域内的相互作用是稳定的，而铰链和血红素之间的相互作用是不稳定的。相反，位点98和99的突变改变咪唑结合动力学，但不改变铰链运动。因此，在铰链区离开血红素后，这些突变似乎影响了细胞色素的结构，导致溶剂对结合的咪唑的接触增加，或者改变了蛋白质与结合的咪唑之间的相互作用。
All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case ofRhodobacter capsulatuscytochrome c2, the sixth heme ligand Met96 and up to 14 flanking residues (positions 88−100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region ofRhodobacter capsulatuscytochrome c2and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93−100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.