Bosshard H R, Bänziger J, Hasler T, Poulos T L
J Biol Chem. 1984 May 10;259(9):5683-90.
The histidine-selective reagent diethyl pyrocarbonate and dye-sensitized photooxidation have been used to study the functional role of histidines in cytochrome c peroxidase. Of the 6 histidines in cytochrome c peroxidase, 5 are modified by diethyl pyrocarbonate at alkaline pH and 4 by photooxidation. The sixth histidine serves as the proximal heme ligand and is unavailable for reaction. Both modification reactions result in the loss of enzymic activity. However, photooxidized peroxidase retains its ability to react with H2O2 and to form a 1:1 cytochrome c peroxidase-cytochrome c complex. It is, therefore, concluded that the extra histidine modified by diethyl pyrocarbonate is the catalytic site distal histidine, His 52. In the presence of cytochrome c, no enzymic activity is lost by photooxidation and a single histidine, His 181, is protected from oxidative destruction. This finding provides strong support for the hypothetical model of the cytochrome c peroxidase-cytochrome c complex in which His 181 lies near the center of the intermolecular interface where it seems to provide an important link in the electron transfer process.
组氨酸选择性试剂焦碳酸二乙酯和染料敏化光氧化已被用于研究组氨酸在细胞色素c过氧化物酶中的功能作用。细胞色素c过氧化物酶中的6个组氨酸中，有5个在碱性pH下被焦碳酸二乙酯修饰，4个被光氧化修饰。第6个组氨酸作为近端血红素配体，不能参与反应。两种修饰反应均导致酶活性丧失。然而，光氧化的过氧化物酶保留了与H2O2反应并形成1:1细胞色素c过氧化物酶-细胞色素c复合物的能力。因此，可以得出结论，被焦碳酸二乙酯修饰的额外组氨酸是催化位点远端组氨酸，即His 52。在细胞色素c存在的情况下，光氧化不会导致酶活性丧失，并且单个组氨酸His 181受到保护不被氧化破坏。这一发现为细胞色素c过氧化物酶-细胞色素c复合物的假设模型提供了有力支持，在该模型中，His 181位于分子间界面的中心附近，似乎在电子传递过程中提供了重要的联系。
