MEASUREMENTS OF CO GEMINATE RECOMBINATION IN CYTOCHROMES P450 AND P420

The kinetics of CO geminate recombination in cytochrome P450(cam) are studied at room temperature subsequent to laser photolysis. The gemina te rebinding kinetics of P450 are strongly affected by the presence of the camphor substrate, We observe a similar to 2% geminate yield for substrate-bound P450 and a 90% geminate yield when the substrate is ab sent. The drastic difference in the geminate kinetics suggests that th e presence of camphor significantly alters the CO rebinding and escape rates by modifying the heme pocket environment. Two geminate phases a nd two bimolecular rebinding phases in the substrate free protein were observed, which could arise from slowly interconverting protein confo rmations. When the temperature or the viscosity of the solution is cha nged, the fast geminate rate remains the same, whereas the slow gemina te rate and the two bimolecular rates change significantly. The gemina te rebinding yield of substrate-free P420 is smaller than that of subs trate free P450, but its geminate rebinding rate is faster, This demon strates that in the absence of substrate, CO escapes from the pocket o f P420 much more rapidly than from P450 and suggests that the distal p ocket environment is altered in the P420 form.