Sono M, Eble K S, Dawson J H, Hager L P
J Biol Chem. 1985 Dec 15;260(29):15530-5.
Extensive spectroscopic investigations of chloroperoxidase and cytochrome P-450 have consistently revealed close similarities between these two functionally distinct enzymes. Although the CO-bound ferrous states were the first to display such resemblance, additional comparisons have focused on the native ferric and ferrous and the ligand-bound ferric derivatives of the enzymes. In order to test the extent to which the spectral properties of the two enzymes match each other, we have prepared the NO, alkyl isocyanide, and O2 adducts of ferrous chloroperoxidase, the latter two for the first time. As expected, the NO adducts of the two proteins have similar UV-visible absorption and magnetic circular dichroism spectra; the same behavior is observed for the alkyl isocyanide complexes. Unexpectedly, the dioxygen adduct of ferrous chloroperoxidase (i.e. Compound III), generated in cryogenic solvents at -30 degrees C by bubbling with O2, is spectrally distinct from oxy-P-450-CAM. Identification of this derivative as oxygenated chloroperoxidase is based on the following criteria: It is EPR-silent at 77 K. The bound O2 is dissociable as judged by the uniform conversion to the CO-bound form. Oxy-chloroperoxidase autoxidizes to form the native ferric enzyme without detectable intermediates at a rate comparable to that determined for oxy-P-450-CAM. Oxy-chloroperoxidase exhibits optical absorption (lambda nm (epsilon mM) = 354 (41), 430 (94), 554 (16.5), 587 (12.5)) and magnetic circular dichroism spectra that are clearly distinct from those of histidine-ligated heme proteins such as oxy-myoglobin or oxy-horseradish peroxidase. Surprisingly, several of its spectral properties, namely the red-shifted Soret peak and discrete alpha peak, are also unlike those of oxy-P-450-CAM. Since considerable evidence has accumulated supporting the ligation of an endogenous thiolate to the heme iron of chloroperoxidase, as has been established for the P-450 enzyme, the observed dissimilarities suggest that the electronic properties of the two dioxygen adducts are quite sensitive to differences in their active site heme environment. This, in turn may be related to the functional differences between the two enzymes.
对氯过氧化物酶和细胞色素P - 450进行的广泛光谱研究一直揭示出这两种功能不同的酶之间存在密切相似性。尽管一氧化碳结合的亚铁状态是最先显示出这种相似性的，但更多的比较集中在酶的天然铁离子和亚铁离子以及配体结合的铁离子衍生物上。为了测试这两种酶的光谱特性相互匹配的程度，我们制备了亚铁氯过氧化物酶的一氧化氮、烷基异氰化物和氧气加合物，后两者是首次制备。正如预期的那样，两种蛋白质的一氧化氮加合物具有相似的紫外可见吸收光谱和磁圆二色光谱；烷基异氰化物配合物也观察到相同的行为。出乎意料的是，通过在 - 30℃的低温溶剂中鼓泡氧气生成的亚铁氯过氧化物酶的双氧加合物（即化合物III）在光谱上与氧合P - 450 - CAM不同。将该衍生物鉴定为氧化氯过氧化物酶基于以下标准：它在77K时电子顺磁共振沉默。结合的氧气可解离，这通过向一氧化碳结合形式的均匀转化来判断。氧合氯过氧化物酶自氧化形成天然铁离子酶，其速率与氧合P - 450 - CAM测定的速率相当，且没有可检测到的中间体。氧合氯过氧化物酶表现出的光吸收（λnm（εmM） = 354（41），430（94），554（16.5），587（12.5））和磁圆二色光谱与组氨酸连接的血红素蛋白如氧合肌红蛋白或氧合辣根过氧化物酶的光谱明显不同。令人惊讶的是，它的几个光谱特性，即红移的Soret峰和离散的α峰，也与氧合P - 450 - CAM不同。由于已经积累了大量证据支持内源性硫醇盐与氯过氧化物酶的血红素铁结合，正如P - 450酶所确定的那样，观察到的差异表明两种双氧加合物的电子性质对其活性位点血红素环境的差异非常敏感。反过来，这可能与两种酶之间的功能差异有关。
