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
