Structural Stability and Dynamics of Hydrogenated and Perdeuterated Cytochrome P450cam (CYP101)
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- 作者：Flora Meilleur ; Jö ; rg Contzen ; Dean A. A. Myles ; and Christiane Jung
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：July 13, 2004
- 年：2004
- 卷：43
- 期：27
- 页码：8744 - 8753
- 全文大小：203K
- 年卷期：v.43,no.27(July 13, 2004)
- ISSN：1520-4995
文摘
Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida,has been characterized concerning thermal stability and structural dynamics. For the first time, Fouriertransform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondarystructure compositions were determined from the fitted amide I' spectral region, giving band populationsat 10 
C for the perdeuterated protein of 22% between 1605 and 1624 cm-1 (
-sheets), 47% between1633 and 1650 cm-1 (
-helix (29%) plus unordered/310-helix (18%)), and 28% between 1657 and 1677cm-1 (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm-1 (-sheets), 52%between 1640 and 1658 cm-1 (-helix (41%) plus unordered/310-helix (11%)), and 24% between 1665and 1680 cm-1 (turns).Thermal unfolding experiments revealed that perdeuterated P450cam was less stablethan the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 C for theperdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to theP450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of theligand occurred with the same kinetics and rate constants kon, 8.9 × 104 and 8.3 × 104 M-1 s-1 for theperdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutroncrystallographic study to determine the specific hydration states and hydrogen-bonding networks at theactive site. The analyses presented here show that perdeuterated P450cam is structurally similar to itshydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained fromthe neutron structure will be representative of the normal hydrogenated P450cam.
C for the perdeuterated protein of 22% between 1605 and 1624 cm-1 (-sheets), 47% between1633 and 1650 cm-1 (-helix (29%) plus unordered/310-helix (18%)), and 28% between 1657 and 1677cm-1 (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm-1 (-sheets), 52%between 1640 and 1658 cm-1 (-helix (41%) plus unordered/310-helix (11%)), and 24% between 1665and 1680 cm-1 (turns).Thermal unfolding experiments revealed that perdeuterated P450cam was less stablethan the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 C for theperdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to theP450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of theligand occurred with the same kinetics and rate constants kon, 8.9 × 104 and 8.3 × 104 M-1 s-1 for theperdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutroncrystallographic study to determine the specific hydration states and hydrogen-bonding networks at theactive site. The analyses presented here show that perdeuterated P450cam is structurally similar to itshydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained fromthe neutron structure will be representative of the normal hydrogenated P450cam.