Hildebrandt P, Greinert R, Stier A, Taniguchi H
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, FRG.
Eur J Biochem. 1989 Dec 8;186(1-2):291-302. doi: 10.1111/j.1432-1033.1989.tb15208.x.
The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form, P-420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P-450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P-450 isozymes.
利用共振拉曼光谱对兔微粒体细胞色素P - 450的同工酶2和4（LM2、LM4）进行了研究。基于高质量光谱，给出了细胞色素P - 450 LM2和LM4不同铁离子状态下，在100 - 1700 cm⁻¹频率范围内卟啉模式的振动归属。共振拉曼光谱根据血红素铁的自旋和配位状态以及血红素 - 蛋白质相互作用进行解释。在细胞色素P - 450 LM2中，主要占据六配位低自旋构型，而在同工酶LM4中，五配位高自旋形式是最稳定的状态。这两种自旋构型在LM2和LM4中不同的稳定性可归因于活性位点的结构。在同工酶LM4的低自旋形式中，蛋白质基质迫使血红素形成比LM2中更刚性的构象。当第六个配体解离时，这些空间限制被消除，导致同工酶LM4高自旋形式的活性位点结构更灵活。发现乙烯基的振动模式是两种同工酶中血红素口袋特定结构的特征标记。它们对I型底物结合也有敏感响应。在细胞色素P - 450 LM4中，底物结合口袋的占据诱导乙烯基的构象变化，这通过乙烯基模式的频率移动反映出来，而在LM2同工酶中，这些取代基的基态构象不受影响，这表明更灵活的血红素口袋可以容纳底物而不对卟啉施加空间限制。共振拉曼技术使底物结合诱导的结构变化可见，并且独立于与自旋状态平衡移动相关的变化：底物结合和未结合底物的酶中的高自旋状态在结构上是不同的。无活性形式P - 420的形成涉及血红素结合口袋中的严重结构重排，导致乙烯基构象的剧烈变化。在这项工作中观察到的细胞色素P - 450 LM2和LM4活性位点的构象差异有助于理解细胞色素P - 450同工酶底物和产物特异性的结构基础。
