Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states.
Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states.
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不同构象状态下细胞色素 C 过氧化物酶-一氧化碳加合物的拉曼光谱和红外光谱。
DOI：
10.1021/bi00363a038
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Spiro,TG
中科院分区：
文献类型：
--
作者：
Smulevich,G;Evangelista-Kirkup,R;English,A;Spiro,TG
1986年3月17日收到的修订版Mandarin pt摘要：报告了CO结合细胞色素c过氧化物酶（CCP）的共振拉曼（RR）光谱。在低pH下，观察到两种形式：形式II，其中i/Fe_c=530 cm-1和dFeCO= 585 cm-1，以及形式I，其中i/Fe_c=495 cm-1和不可检测的5 FeCO。它们在1922 cm-1处有重合的红外波段。这些低pH值的形式，类似于辣根过氧化物酶（HRP）所观察到的，是由于倾斜，氢键合的CO和垂直CO，分别。两种蛋白质的频率不同，CCP与CO的氢键较弱。与HRP，形式I和II之间的平衡向后者转移，在增加CO浓度，这表明CO的二次结合扰乱远端残基。在高pH值[8.4，三（羟甲基）aminomethanebuffer]的形式II部分转化为另一种形式，IT，与v？eC= 503 cm-1，6 FeCO = 575 cm-1，和eC = 1948 cm-1;提出了倾斜的非H键合的几何结构。然而，如果使用磷酸盐缓冲液，则形式II（H键合）在pH 8.4下持续存在。这一结果确立了磷酸盐使酶的氢键形式不稳定的作用;这表明磷酸盐在远端咪唑附近结合并显著增加其pKa。构象状态也受到老化的影响。如高频RR光谱所监测，新鲜蛋白质含有纯粹的高自旋Fe 111血红素，并且几乎只在CO浓度升高时产生II型。老化的蛋白质显示出大量的低自旋Fe 111组分和大部分的形式I，即使在升高的CO浓度。此外，II型-* IF型转化的磷酸盐抑制作用在老化时丧失。CO结合的模型提出，其中涉及的远端残基的替代构象。这种平衡受到质子、磷酸盐和第二个CO分子结合的影响，并且对衰老时发生的不可逆蛋白质变化敏感。
Revised Manuscript Received March 17, 1986 abstract: Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with i/Fe_c=530 cm" 1 and dFeCO= 585 cm" 1, and form I, with i/Fe_c=495 cm" 1 and no detectable 5FeC0. They appearto have coincident vco infrared bands, at 1922 cm" 1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicatedfor CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris (hydroxymethyl) aminomethanebuffer] the form II fraction converts to another form, IT, with v? eC= 503 cm" 1, 6FeCO= 575 cm" 1, and vco= 1948 cm" 1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate instabilizing the H-bonded form of theenzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its p. Ka. The conformational state is also influenced by aging. Fresh protein contains purely high spin Fe111 heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations. Aged protein showed a substantial low-spin Fe111 component and a large fraction of form I, even at elevated CO concentration. In addition, the phosphate inhibition of the form II-* form IF conversion is lost upon aging. A model for CO binding is put forward, which involves alternative conformations of the distal residues. The equilibrium is influenced by the binding of protons, phosphate, and a second CO molecule and is sensitive to irreversible protein changes that occur upon aging.