Resonance Raman Spectral Properties and Stability of Manganese Protoporphyrin IX Cytochrome b 5

• Published in: Biochemistry (Easton) Vol. 36; no. 23; pp. 7114 - 7125
• Main Authors: Gruenke, Larry D, Sun, Jie, Loehr, Thomas M, Waskell, Lucy
• Format: Journal Article
• Language: English
• Published: American Chemical Society 10.06.1997
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi970407p

The structure and stability of cytochrome b 5 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 b 5 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 b 5 indicated a high-spin pentacoordinate structure which was independent of laser power. Studies of the binding of MnIII protoporphyrin IX to apocytochrome b 5 indicated that the MnIII-containing porphyrin bound much less tightly to the protein than did heme. Although the second-order rate constant at 20 °C for the association of heme with apocytochrome b 5 (4.5 × 107 M-1 s-1) was estimated to be only 1 order of magnitude higher than that with Mn protoporphyrin IX (3.3 × 106 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 × 10-2 s-1 at 20 °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 × 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 b 5 increased with temperature from 4 nM at 20 °C to 14 nM at 37 °C. These results suggest that, in the reconstituted cytochrome P450 metabolizing system, especially in studies done with low protein concentrations (0.1 μM), and at elevated temperatures (37 °C), as much as 30% of the manganese-substituted cytochrome b 5 may dissociate to free Mn−protoporphyrin IX and apocytochrome b 5.