Dynamics of Electron Transport in Cytochrome P450 Systems Studied at Sub-Zero Temperature

Experimentation in fluid mixed solvents (1 : 1 v/v phosphate buffer ethylene glycol) at sub-zero temperatures has permitted us to record the two univalent reductions of the bacterial cytochrome P450 by the natural electron donor putidaredoxin, without recycling or alternative pathway reactions. Dynamic evidence shows the formation of putidaredoxincytochrome complexes prior to electron transfer. The complex formation is rate limiting in the first reduction and in our experimental conditions. The kinetics of binding between the two oxidized proteins has also been recorded in the same medium under various conditions of concentration, temperature and ionic strength. At very low ionic strength, the rate is limited by electrostatic repulsion between the two negatively charge proteins; above I = 0.03 this effect appears negligible and the affinity seems to be governed by hydrophobic interaction free energy