The structure and stability of cytochrome b5 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 MnIII cytochrome b5 was consistent with a high-spin hexacoordinate MnIII protoporphyrin IX structure that converted to a high-spin pentacoordinate structure at higher laser power. The resonance Raman spectrum of MnII cytochrome b5 indicated a high-spin pentacoordinate structure which was independent of laser power. Studies of the binding of MnIII protoporphyrin IX to apocytochrome b5 indicated that the MnIII-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 b5 (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 b5 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 b5 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 b5 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 microM), and at elevated temperatures (37 degrees C), as much as 30% of the manganese-substituted cytochrome b5 may dissociate to free Mn-protoporphyrin IX and apocytochrome b5.

译文

用共振原拉曼光谱和停止流可见光谱研究了用锰原卟啉IX代替铁原卟啉IX重构的细胞色素b5的结构和稳定性。 MnIII细胞色素b5的共振拉曼光谱与高自旋六配位MnIII原卟啉IX结构一致,后者在更高的激光功率下转换为高自旋五配位结构。 MnII细胞色素b5的共振拉曼光谱表明高自旋五坐标结构,与激光功率无关。 MnIII原卟啉IX与脱细胞色素b5结合的研究表明,含MnIII的卟啉与蛋白质的结合比血红素少得多。虽然血红素与脱细胞色素b5(4.5 x 10(7)M(-1)s(-1))的结合在20摄氏度的二阶速率常数估计仅比其高1个数量级锰原卟啉IX(3.3 x 10(6)M(-1)s(-1)),锰取代的细胞色素b5分解为载脂蛋白和游离Mn原卟啉IX的一级速率常数为1.2 x 10( -2)在20摄氏度时为s(-1),而室温下血红素从细胞色素b5的解离发生的速度要慢3个数量级,其一阶速率常数为1.67 x 10(-5)s(-1) [Vergeres,G.,Chen,DY,Wu,FF,&Waskell,L.(1993)Arch。生化。生物物理学。 305,231-241]。锰取代的细胞色素b5的平衡解离常数随温度从20摄氏度的4 nM增加到37摄氏度的14 nM。 0.1 microM)和在升高的温度(37摄氏度)下,多达30%的锰取代的细胞色素b5可能解离成游离的Mn-原卟啉IX和载脂细胞色素b5。

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