Isothermal titration calorimetric studies on the associations of putidaredoxin to NADH-putidaredoxin reductase and P450cam.
Putidaredoxin (Pdx), an iron-sulfur protein containing a 2Fe-2S cluster, serves as a physiological electron mediator from NADH-putidaredoxin reductase (PdR) to P450cam in the P450cam monooxygenation reaction cycle. Previous studies have revealed that the associations of Pdx with P450cam and PdR are not strongly dominated by electrostatic interactions, although such interactions stabilize most electron-transfer complexes [A.R. De Pascalis, I. Jelesarov, F. Ackermann, W.H. Koppenol, M. Hiroasawa, D.B. Knaff, H.R. Bosshard, Protein Sci. 2 (1993) 1126-1135]. In the present study, to elucidate the interactions dominating the specific associations in the electron-transfer reaction mediated by Pdx, the thermodynamic properties--entropy (delta S), enthalpy (delta H), and heat capacity changes (delta Cp)--for PdR/Pdx and P450cam/Pdx association reactions have been examined by isothermal titration calorimetry (ITC). Although the binding enthalpy change, delta Hbind, for the PdR/Pdx association is positive at 10 degrees C, it declines linearly with temperature in the range 10-22 degrees C and becomes negative above 11 degrees C. On the other hand, the binding entropy change, delta Sbind, is positive at all temperatures examined in this study, indicating that the association of Pdx to PdR is entropically driven. On the basis of the temperature dependence of delta Hbind, delta Cpbind for the association of Pdx to PdR was estimated as -1.24 kJ mol-1 K-1. This value is larger than those reported for other electron-transfer protein systems (e.g., -0.68 kJ mol-1 K-1 for ferredoxin/ferredoxin: NADP+ reductase), suggesting that the PdR/Pdx association may be dominated by hydrophobic rather than electrostatic components. For the P450cam/Pdx association, the negative delta Sbind and highly favorable delta Hbind were observed, behavior that stands in sharp contrast to the association reactions in other electron-transfer proteins. The energetics of the P450cam/Pdx association are similar to those of binding reaction of antibody to antigen in which van der Waals and hydrogen bonding interactions are dominant, resulting in high specificity in the association of Pdx with P450cam.
Aoki
M
M
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Ishimori
K
K
Fukada
H
H
Takahashi
K
K
Morishima
I
I
D000818:Animals; D002151:Calorimetry; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism
D005288:Ferredoxins; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase