Abstract
The kinetics of CO geminate recombination in cytochrome P450cam are studied at room temperature subsequent to laser photolysis. The geminate rebinding kinetics of P450 are strongly affected by the presence of the camphor substrate. We observe a approximately 2% geminate yield for substrate-bound P450 and a 90% geminate yield when the substrate is absent. The drastic difference in the geminate kinetics suggests that the presence of camphor significantly alters the CO rebinding and escape rates by modifying the heme pocket environment. Two geminate phases and two bimolecular rebinding phases in the substrate free protein were observed, which could arise from slowly interconverting protein conformations. When the temperature or the viscosity of the solution is changed, the fast geminate rate remains the same, whereas the slow geminate rate and the two bimolecular rates change significantly. The geminate rebinding yield of substrate-free P420 is smaller than that of substrate free P450, but its geminate rebinding rate is faster. This demonstrates that in the absence of substrate, CO escapes from the pocket of P420 much more rapidly than from P450 and suggests that the distal pocket environment is altered in the P420 form.
Highlights
Carmelo Di Primo, Nancy Gerber, and Stephen G
The geminate rebinding yield of substrate-free P420 is smaller than that of substrate free P450, but its geminate rebinding rate is faster. This demonstrates that in the absence of substrate, CO escapes from the pocket of P420 much more rapidly than from P450 and suggests that the distal pocket environment is altered in the P420 form
In the ferric state of P420, H 20 remains as a heme ligand, whereas alterations of the protein tertiary structure lead to a significant reduction in affinity for Cys357 thiolate binding to the heme iron
Summary
P450m08 was generated and purified from P. putida as described previously [28]. High purity P420m0 ' was prepared by exposure of P450m"' to a pressure of kbar at room temperature for 1-2 h [29, 30]. Due to the limited time resolution of the instruments, kinetics faster than 10 ns are not observed This leads to 10-20% "missing" amplitude, as determined by the expected equilibrium absorption change (M(O)) between P450m::00 and P450mr at the probe wavelength. It should be noted that this methodology does not account for the possibility of spectral evolution caused by nonequilibrium protein relaxation [19]; such effects are expected to be relatively small (-25%). Another source of uncertainty in the geminate kinetics arises from radiofrequency noise from the Nd:YAG laser. The remaining noise still affects the accuracy of the measurement at the level of :S 1% near 10- 7 s
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