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 microM 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 (ELUMO) of the resorufin O-anion is higher than the ELUMO value for the protonated form. This low ELUMO 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.(ABSTRACT TRUNCATED AT 250 WORDS)[1]References
- Experimental and theoretical study on the redox cycling of resorufin by solubilized and membrane-bound NADPH-cytochrome reductase. Balvers, W.G., Boersma, M.G., Vervoort, J., Rietjens, I.M. Chem. Res. Toxicol. (1992) [Pubmed]
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