CO binding and ligand discrimination in human myeloperoxidase.
CO binding and ligand discrimination in human myeloperoxidase.
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DOI：
10.1021/bi9021507
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发表时间：
2010-03-16
期刊：
影响因子：
2.9
通讯作者：
Rich PR
中科院分区：
文献类型：
--
作者：
Murphy EJ;Maréchal A;Segal AW;Rich PR
尽管铁髓过氧化物酶（MPO）可以结合O2和NO，但其结合CO的能力受到质疑。紫外/可见光谱法用于证实CO会引起二价MPO的微小光谱位移，而傅里叶变换红外差谱法明确表明这些位移是由亚铁血红素-CO化合物的形成引起的。在618和645 nm处监测CO光解后的分解速率，作为CO浓度和pH的函数。在pH 6.3时，kon和koff分别为0.14 mM−1·s−1和0.23 s−1，产生异常高的KD 1.6 mM。MPO对CO的亲和力比对O2的亲和力弱10倍。所观察到的CO结合的速率常数随pH值的增加而增加，并由一个单一的质子化基团与pKa为7.8。傅立叶变换红外光谱显示了两种不同的结合CO的构象，频率分别为1927和1942 cm−1。它们的重组速率常数是相同的，这表明两种形式的结合CO是在快速热平衡，而不是两个不同的蛋白质群体具有不同的结合位点。结合状态的比例是pH值依赖性的（pKa ≥ 7.4）与1927 cm−1的形式有利于在高pH值。结构因素，占MPO的配体结合特性的确定与其他配体结合血红素蛋白的范围内发表的数据进行比较，并支持最近的建议，即在MPO的近端His 336是在一个真正的咪唑状态。
Despite the fact that ferrous myeloperoxidase (MPO) can bind both O2 and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous–CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, kon and koff were 0.14 mM−1·s−1 and 0.23 s−1, respectively, yielding an unusually high KD of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O2. The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pKa of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm−1. Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pKa ≈ 7.4) with the 1927 cm−1 form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state.