Denisov Ilia G, Mak Piotr J, Makris Thomas M, Sligar Stephen G, Kincaid James R
Department of Biochemistry, University of Illinois, Urbana, Illinois 61801, USA.
J Phys Chem A. 2008 Dec 18;112(50):13172-9. doi: 10.1021/jp8017875.
Resonance Raman (RR) studies of intermediates generated by cryoreduction of the oxyferrous complex of the D251N mutant of cytochrome P450(cam) (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. In as much as previous EPR and ENDOR studies have shown that annealing of this species to approximately 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 nu(O-O) mode decreases from a superoxo-like frequency near approximately 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 heme-bound peroxide formulation. This species also exhibits a nu(Fe-O) mode, the 553 cm(-1) frequency of which is higher than that observed for the nonreduced 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 nu(O-O) mode at 774 cm(-1), whereas the nu(Fe-O) increases to 564 cm(-1). Both modes exhibit a downshift 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.
报道了对细胞色素P450（cam）（CYP101）的D251N突变体的氧亚铁配合物进行低温还原所产生中间体的共振拉曼（RR）研究。由于该突变体中质子向活性位点的传递受阻，在77 K的冷冻溶液中对氧配合物进行辐射还原后，未质子化的过氧铁中间体被观察为主要物种。鉴于先前的电子顺磁共振（EPR）和电子核双共振（ENDOR）研究表明，将该物种加热到约180 K会导致远端氧原子质子化形成氢过氧中间体，因此利用该体系直接通过RR研究还原及随后质子化引起的Fe - O和O - O键的变化。我们的结果表明，还原后ν（O - O）模式从接近约1130 cm⁻¹的超氧类似频率降至792 cm⁻¹。后一频率及其对H/D交换的不敏感性与血红素结合的过氧化物结构一致。该物种还表现出ν（Fe - O）模式，其553 cm⁻¹的频率高于未还原氧合P450前体所观察到的频率（537 cm⁻¹），这意味着还原后Fe - O键增强。随后质子化时，生成的Fe - O - OH片段在774 cm⁻¹处表现出降低的ν（O - O）模式，而ν（Fe - O）增加到564 cm⁻¹。如氢过氧铁结构所预期的那样，两种模式在H/D交换时均发生下移。将这些实验RR数据与先前为野生型蛋白获得的数据进行比较，并参照理论预测讨论还原及随后质子化时观察到的位移。
