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首页> 外文期刊>Biochemistry >Identification of Proximal and Distal Axial Ligands in Leishmania major Pseudoperoxidase
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Identification of Proximal and Distal Axial Ligands in Leishmania major Pseudoperoxidase

机译：利什曼原虫主要伪过氧化物酶中近端和远端轴配体的鉴定

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Previous optical and electron paramagnetic resonance (EPR) spectroscopic studies of the newly discovered peroxynitrite scavenging pseudoperoxidase from Leishmania major (LmPP) suggested that ferric LmPP contained a six-coordinate low-spin (6cLS) heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. To identify the axial ligands of LmPP, we exploit a systematic mutational analysis of potential heme ligands. On the basis of UV?visible and EPR spectroscopy, we report that the substitution of the proximal His206 with alanine in LmPP alters the 6cLS to a five-coordinate high spin (5cHS) form at pH 4.0 that has a spectrum characteristic of a Cys-ligated 5cHS derivative. The electronic absorption and EPR analysis of all alanine-substituted Cys and Met single mutants establish that when Cys107 is replaced with alanine, a new species appears that has a spectrum characteristic of a histidine-ligated 5cHS derivative at pH 4.0. Together, these results suggest that His206 and Cys107 act as the proximal and distal axial ligands in ferric LmPP, respectively. However, the electronic properties of reduced wild-type LmPP are similar to those of known 5cHS His-ligated heme proteins at pH 8.8, indicating that the thiolate bond was broken upon reduction. Furthermore, the wild-type protein was only partially reduced at pH 4.0, but the E105L mutant was completely reduced to form a 5cHS ferrous heme. These results imply that the presence of an acidic residue near the distal site may prevent reduction of the heme iron at acidic pH.
机译：先前对来自利什曼原虫病（LmPP）的新发现的过氧亚硝酸盐清除假过氧化物酶的光学和电子顺磁共振（EPR）光谱研究表明，三价铁LmPP包含六坐标低自旋（6cLS）血红素，与硫醇盐配体（大概是半胱氨酸）结合其血红素铁。为了鉴定LmPP的轴向配体，我们利用了潜在的血红素配体的系统突变分析。根据紫外可见光谱和EPR光谱，我们报道在LmPP中用丙氨酸替代近端His206将6cLS改变为在pH 4.0时具有Cys-光谱特征的五配位高自旋（5cHS）形式。连接5cHS衍生物。对所有丙氨酸取代的Cys和Met单个突变体的电子吸收和EPR分析表明，当Cys107替换为丙氨酸时，出现了一个新物种，其在pH 4.0时具有组氨酸连接的5cHS衍生物的光谱特征。总之，这些结果表明，His206和Cys107分别充当LmPP铁的近端和远端轴向配体。但是，还原的野生型LmPP的电子性质与已知的5cHS His连接的血红素蛋白在pH 8.8时的电子性质相似，表明硫醇键在还原时被破坏。此外，野生型蛋白质在pH 4.0下仅部分还原，但E105L突变体被完全还原形成5cHS亚铁血红素。这些结果暗示在远端部位附近存在酸性残基可以防止血红素铁在酸性pH下的还原。

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