Hildebrandt P, Garda H, Stier A, Bachmanova G I, Kanaeva I P, Archakov A I
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, FRG.
Eur J Biochem. 1989 Dec 8;186(1-2):383-8. doi: 10.1111/j.1432-1033.1989.tb15220.x.
The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.
通过对溶液中单体和寡聚体蛋白进行共振拉曼光谱分析，研究了蛋白质 - 蛋白质相互作用和底物结合对兔肝微粒体细胞色素P - 450 LM2活性位点结构的影响。同时比较了两种状态下H2O2依赖性催化活性。血红素的两个乙烯基取代基表现出不同的取向，这由它们伸缩振动的频率和强度表明。一组位于血红素平面内，在细胞色素P - 450 LM2的两种状态下保持不变，另一组则倾斜出平面。寡聚体中的倾斜角度小于单体中的倾斜角度。发现这些乙烯基伸缩模式与一些卟啉模式一起是四级结构和底物结合的敏感指标。在寡聚体和单体中，底物结合都会导致一些卟啉模式和乙烯基伸缩振动的相对强度发生变化，这可能反映了由于结合底物与血红素之间的疏水相互作用引起的电子跃迁修饰。与单体细胞色素P - 450 LM2不同，苄非他明与该同工酶的寡聚体结合还会产生自旋态平衡的移动。这表明在寡聚体中，底物结合口袋通过蛋白质 - 蛋白质相互作用转变为一种结构，迫使底物干扰第六个配体，导致五配位高自旋构型增加。在单体中，底物结合口袋可以容纳苄非他明而不影响自旋态。咪唑与单体和寡聚体细胞色素P - 450 LM2的结合产生基本相同的共振拉曼光谱。显然，用咪唑取代天然的第六个配体会以一种使其对蛋白质 - 蛋白质相互作用不敏感的方式扰乱活性位点的结构。细胞色素P - 450 LM2对苄非他明的H2O2依赖性N - 去甲基化和苯胺对羟基化不依赖于其聚集状态。
