McLean M A, Yeom H, Sligar S G
Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana 61801, USA.
Biochimie. 1996;78(8-9):700-5. doi: 10.1016/s0300-9084(97)82527-8.
The kinetics of carbon monoxide binding to cytochrome P450BM-3 in the presence and absence of substrate has been investigated using flash photolysis. The second order kinetics for CO association with the substrate-free form of the protein appear biphasic. Deconvolution into two exponentials yields fast and slow rate constants of 11.1 +/- 0.6 x 10(6) M-1 s-1 and 3.5 +/- 0.2 x 10(6) M-1 s-1, respectively with 52% of the signal being attributed to the fast phase. Interestingly, upon binding of a substrate such as laurate, the second order kinetics become monophasic, with a value of 3.5 x 10(6) M-1 s-1, which are similar to the slow rate found in the substrate-free form of the protein. We have also examined the geminate CO rebinding kinetics in the presence and absence of various substrates. In the substrate-free form of the overall geminate yield is 30%, and addition of a substrate increases the geminate yield to roughly 50%. Both the substrate-free and substrate-bound forms exhibit complex geminate kinetics which cannot be described by a simple three-state kinetic model. Extension of this model to include four states is required. The addition of substrate causes an increase in the geminate rate constants resulting in a larger geminate amplitude when compared to the substrate-free form. There is also evidence for a correlation between the volume occupied by the substrate and the geminate rate constants. These results are discussed in terms of substrate-dependent conformational changes in cytochrome P450BM-3 and the overall energy landscape of the hemoprotein which couples to conformer equilibria.
