Resonance Raman spectral properties and stability of manganese protoporphyrin IX cytochrome b(5)
Resonance Raman spectral properties and stability of manganese protoporphyrin IX cytochrome b(5)
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DOI：
10.1021/bi970407p
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发表时间：
1997-06-10
期刊：
影响因子：
2.9
通讯作者：
Waskell, L
中科院分区：
文献类型：
--
作者：
Gruenke, LD;Sun, J;Waskell, L
用共振拉曼光谱和停流可见光谱研究了锰原卟啉IX代替铁原卟啉IX重组细胞色素b的结构和稳定性。Mn-III细胞色素b的共振拉曼光谱与高自旋六配位Mn-III原卟啉IX结构一致，该结构在较高的激光功率下转化为高自旋五配位结构。Mn-Ⅱ细胞色素B（5）的共振拉曼光谱表明其具有高自旋五配位结构，与激光功率无关。对Mn-Ⅲ原卟啉IX与脱辅基细胞色素B（5）-结合的研究表明，含Mn-Ⅲ的卟啉与蛋白质的结合比血红素要紧密得多。虽然血红素与脱辅基细胞色素B（5）结合的二级速率常数在20 ℃时（4.5 × 10（7）M-1 s（-1））的浓度仅比Mn原卟啉IX高1个数量级（3.3 x 10（6）M-1 s（-1）），锰取代的细胞色素B（5）解离成脱辅基蛋白和游离的锰原卟啉IX，在20 ℃时的一级速率常数为1.2 × 10（-2）s（-1），而在室温下血红素从细胞色素B（5）中的解离要慢3个数量级，一级速率常数为1.67 × 10（-5）s（-1）[Vergeres，G.，Chen，中国粘蝇D.是的，Wu，F. F.，& Waskell，L.（1993）Arch，Biochem. Biophys. 305，231-241]。锰取代的细胞色素b的平衡解离常数随温度从20 ℃的4 nM增加到37 ℃的14 nM。这些结果表明，在重组细胞色素P450代谢系统中，特别是在低蛋白浓度（0.1 μ M）和高温（37 ℃）下进行的研究中，多达30%的锰取代细胞色素B（5）可能解离为游离的锰-原卟啉IX和脱辅基细胞色素B（5）。
The structure and stability of cytochrome bs reconstituted with manganese protoporphyrin IX instead of iron protoporphyrin IX has been investigated by resonance Raman spectroscopy and stopped-flow visible spectroscopy. The resonance Raman spectrum of Mn-III cytochrome bs was consistent with a high-spin hexacoordinate Mn-III protoporphyrin IX structure that converted to a high-spin pentacoordinate structure at higher laser power. The resonance Raman spectrum of Mn-II cytochrome b(5) indicated a high-spin pentacoordinate structure which was independent of laser power. Studies of the binding of Mn-III protoporphyrin IX to apocytochrome b(5)- indicated that the Mn-III-containing porphyrin bound much less tightly to the protein than did heme. Although the second-order rate constant at 20 degrees C for the association of heme with apocytochrome b(5) (4.5 x 10(7) M-1 s(-1)) was estimated to be only 1 order of magnitude higher than that with Mn protoporphyrin IX (3.3 x 10(6) M-1 s(-1)), the dissociation of manganese substituted cytochrome b(5) into the apoprotein and free Mn protoporphyrin IX occurs with a first-order rate constant of 1.2 x 10(-2) s(-1) at 20 degrees C while the dissociation of heme from cytochrome b(5) at room temperature occurs 3 orders of magnitude more slowly with a first-order rate constant of 1.67 x 10(-5) s(-1) [Vergeres, G., Chen, D. Y., Wu, F.F., & Waskell, L. (1993) Arch, Biochem. Biophys. 305, 231-241]. The equilibrium dissociation constant for manganese substituted cytochrome bs increased with temperature from 4 nM at 20 degrees C to 14 nM at 37 degrees C. These results suggest that, in the reconstituted cytochrome P450 metabolizing system, especially in studies done with low protein concentrations (0.1 mu M), and at elevated temperatures (37 degrees C), as much as 30% of the manganese-substituted cytochrome b(5) may dissociate to free Mn-protoporphyrin IX and apocytochrome b(5).