EXPERIMENTAL AND THEORETICAL-STUDY ON THE REDOX CYCLING OF RESORUFIN BY SOLUBILIZED AND MEMBRANE-BOUND NADPH CYTOCHROME REDUCTASE

The present study describes both experimental and theoretical data on the redox cycling of resorufins catalyzed by NADPH-cytochrome reductase. At 1-5-mu-M concentrations at physiological pH, the redox cycling of ethoxy- and pentoxyresorufin was shown to be far more efficient than the redox cycling of their product from the cytochrome P-450 dependent O-dealkylation, resorufin (7-hydroxyphenoxazone). This was shown to result from the fact that (i) the protonated form of the resorufin is a much better substrate for redox cycling than the deprotonated resorufin O-anion and (ii) at physiological pH the redox cycling active protonated form is present at only 1-4% of the total amount of resorufin. In addition to experimental data, AM1 molecular orbital computer calculations provided evidence for the difference in redox cycling capacity between the resorufin O-anion and its protonated form. The energy of the lowest unoccupied molecular orbital (E(LUMO)) of the resorufin O-anion is higher than the E(LUMO) value for the protonated form. This low E(LUMO) value of the protonated form can be taken as a parameter for its easier reduction. Furthermore, computer calculations demonstrated one-electron reduction of the protonated form to be energetically favorable by 363.5 kJ/mol, compared to one-electron reduction of the deprotonated O-anionic form. Additional AM1 molecular orbital computer calculations indicated that the one-electron-reduced resorufin will become protonated at the O-atom of the intramolecular semiquinone imine moiety before reduction by a second electron becomes likely. Finally, redox cycling of resorufin by solubilized and membrane-incorporated NADPH-cytochrome reductase provided evidence that membrane surroundings increase the concentration of the protonated form of resorufin. This effect is achieved either by favored partitioning of the protonated resorufin into the membrane and/or by an effect of the membrane on the protonation equilibrium of resorufin in favor of the protonated form.