Resonance Raman characterization of the peroxo and hydroperoxo intermediates in cytochrome P450.
Resonance Raman characterization of the peroxo and hydroperoxo intermediates in cytochrome P450.
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DOI：
10.1021/jp8017875
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发表时间：
2008-12-18
期刊：
影响因子：
--
通讯作者：
Kincaid JR
中科院分区：
文献类型：
--
作者：
Denisov IG;Mak PJ;Makris TM;Sligar SG;Kincaid JR
本文报道了细胞色素P450 cam（CYP 101）D251 N突变体的含氧亚铁络合物低温还原产生的中间产物的共振拉曼（RR）研究。由于质子传递到活性位点的事实是在这个突变体中受阻，未质子化的过氧铁中间体被观察到作为主要物种后，在77 K的冷冻溶液中的氧复合物的辐射还原。由于先前的EPR和ENDOR研究已经表明，退火的这种物种的~180 K的结果在质子化的远端氧原子，形成氢过氧中间体，该系统已被利用，以允许直接RR询问的变化，在Fe-O和O-O键所造成的还原和随后的质子化。我们的结果表明，ν（O-O）模在还原时从约1130 cm−1附近的“superoxo类”频率降低到792 cm−1。后者的频率，以及其缺乏H/D交换的敏感性，是符合血红素结合过氧化物配方。该物种还表现出ν（Fe-O）模式，其553 cm−1的频率高于未还原的氧P450前体（537 cm−1），这意味着还原时Fe-O键的增强。在随后的质子化后，所得的Fe-O-OH片段在774 cm−1处显示出降低的ν（O-O）模式，而ν（Fe-O）增加到564 cm−1，这两种模式在H/D交换时显示出下移，正如对氢过氧铁制剂所预期的那样。这些实验RR数据与以前获得的野生型蛋白质和减少和随后的质子化后观察到的变化进行了讨论，参考理论预测。
Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450cam (CYP101) are reported. Owing to the fact that proton delivery to the active site is hindered in this mutant, the unprotonated peroxo-ferric intermediate is observed as the primary species after radiolytic reduction of the oxy-complex in frozen solutions at 77 K. Inasmuch as previous EPR and ENDOR studies have shown that annealing of this species to ~180 K results in protonation of the distal oxygen atom to form the hydroperoxo intermediate, this system has been exploited to permit direct RR interrogation of the changes in the Fe-O and O-O bonds caused by the reduction and subsequent protonation. Our results show that the ν(O-O) mode decreases from a “superoxo-like” frequency near ~1130 cm−1 to 792 cm−1 upon reduction. The latter frequency, as well as its lack of sensitivity to H/D exchange, is consistent with a heme-bound peroxide formulation. This species also exhibits a ν(Fe-O) mode, whose 553 cm−1 frequency is higher than that observed for the non-reduced oxy P450 precursor (537 cm−1), implying a strengthened Fe-O linkage upon reduction. Upon subsequent protonation, the resulting Fe-O-OH fragment exhibits a lowered ν(O-O) mode at 774 cm−1, while the ν(Fe-O) increases to 564 cm−1, both modes exhibiting downshifts upon H/D exchange, as expected for a hydroperoxo-ferric formulation. These experimental RR data are compared with those previously acquired for the wild-type protein and the shifts observed upon reduction and subsequent protonation are discussed with reference to theoretical predictions.
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影响因子：
15
作者：
BAJDOR, K;KINCAID, JR;NAKAMOTO, K
通讯作者：
NAKAMOTO, K
影响因子：
15
作者：
Ibrahim, M;Denisov, IG;Sligar, SG
通讯作者：
Sligar, SG
影响因子：
15
作者：
Davydov, R;Macdonald, IDG;Hoffman, BM
通讯作者：
Hoffman, BM
DOI：
10.1073/pnas.98.2.479
发表时间：
2001-01-16
影响因子：
11.1
作者：
Das, TK;Couture, M;Rousseau, DL
通讯作者：
Rousseau, DL
影响因子：
15
作者：
Davydov, R;Kofman, V;Hoffman, BM
通讯作者：
Hoffman, BM