title: Experimental and theoretical study on the redox cycling of resorufin by solubilized and membrane-bound NADPH-cytochrome reductase.
abstract: 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)
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: W G
authors.initials: WG
authors.lastname: Balvers
authors.forename: M G
authors.initials: MG
authors.lastname: Boersma
authors.forename: J
authors.initials: J
authors.lastname: Vervoort
authors.forename: I M
authors.initials: IM
authors.lastname: Rietjens
mesh_terms: D000818:Animals; D003198:Computer Simulation; D005260:Female; D006863:Hydrogen-Ion Concentration; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D010078:Oxazines; D009249:NADP; C014180:resorufin; D009251:NADPH-Ferrihemoprotein Reductase