Structural and spectroscopic characterization of P450BM3 mutants with unprecedented P450 heme iron ligand sets - New heme ligation states influence conformational equilibria in P450BM3
Structural and spectroscopic characterization of P450BM3 mutants with unprecedented P450 heme iron ligand sets - New heme ligation states influence conformational equilibria in P450BM3
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DOI：
10.1074/jbc.m607949200
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发表时间：
2007-01-05
影响因子：
4.8
通讯作者：
Munro, Andrew W.
中科院分区：
文献类型：
--
作者：
Girvan, Hazel M.;Seward, Harriet E.;Munro, Andrew W.
通过黄细胞色素P450 BM 3血红素结构域的定向诱变产生两个新的P450血红素铁配体组。A264 H和A264 K变体产生Cys-Fe-His和Cys-Fe-Lys轴向配体组，其在结构上得到验证并通过光谱分析表征。EPR和磁性圆二色性（MCD）提供的指纹定义这些P450配体集。近红外MCD光谱确定铁低自旋电荷转移带诊断的新的配体。对于A264 K突变体，这是Cys-Fe-Lys近红外MCD带的首次报道。晶体结构测定表明，无底物的A264 H和A264 K蛋白结晶在不同的构象，如先前观察到的无底物和脂肪酸结合的野生型P450的形式，分别。这反过来可能反映了血红素铁配体的最佳构型所需的蛋白质的Ia螺旋部分的定位。在A264 H晶体的不对称单元中的单体之一是在一个新的构象与一个更开放的基板访问路线的活性位点。相同的物种被分离的野生型血红素结构域，并代表了一种新的构象状态的BM 3（称为SF 2）。这些不同构象的“锁定”从内源性配体不能被底物或外源性配体置换的事实是显而易见的。血红素结构域的构象异质性的随之而来的减少将是重要的尝试，以确定原子结构的全长，多结构域flavocytochrome，从而了解其血红素和还原酶结构域之间的原子细节的相互作用。
Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I a helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.