Ortho-quinone-enhanced ascorbate oxidation. Combined roles of lipid charge and the magnesium cation

Abstract

Quinones are widely distributed compounds in nature. Of these, ortho-quinones are found to be involved in the pathogenic mechanism of Parkinson's disease, in oxidative deaminations to free-radical redox reactions, and as intermediates in the pathways implicated in the carcinogenicity of 2,3- and 3,4-catechol estrogens. Addition of MgCl(2) to solutions of the hydrophobic ortho-quinones, 1,10-phenanthroquinone (PHQ) and beta-lapachone (LQ) enhances ascorbate oxidation in the absence or presence of large unilamellar vesicles (LUVs) of the neutral lipid dimyristoylphos-phatidylcholine (DMPC), although initial rates of ascorbate oxidation are smaller in the presence of lipid as compared to its absence. Addition of this salt to solutions of the para-quinone 1,4-naphthoquinone (NQ) did not affect the ascorbate rate of oxidation in the absence or presence of DMPC. Addition of MgCl(2) to semiquinone solutions of PHQ or LQ in the presence or absence of DMPC increases semiquinone stability, as detected from the semiquinone disproportionation equilibrium displacement to semiquinone formation. Furthermore, MgCl(2) increases the partition of the ortho-semiquinones into the aqueous phase, although no such effect is observed for the semiquinone of NQ. For all the quinones under study, smaller rates of ascorbate oxidation and of semiquinone equilibrium concentration occur in the presence of negatively charged LUVs composed of an equimolar mixture of DMPC and dimyristoylphosphatidic acid DMPA. Ascorbate oxidation rate enhancements correlate with an increase in semiquinone concentration with addition of MgCl(2), in the absence or presence of neutral lipid. This observation favors the proposition that ascorbate oxidation rate increases are caused by semiquinone thermodynamic stabilization. Thus, the ascorbate oxidation rate enhancement by MgCl(2) in solutions containing hydrophobic ortho-quinones is still possible in systems with hydrophobic environments analogous to that of DMPC.

Figure 1

Quinones studied in this work.

Figure 2

Oxygen consumption traces at 37°C of solutions containing 50 mM cacodylate (pH 7.4), 1.0 mM ascorbate and (1) quinone and MgCl₂, (2) quinone and NaCl, (3) quinone, MgCl₂ and DMPC, (4) quinone, NaCl and DMPC, (5) quinone, MgCl₂ and DMPC/DMPA, (6) NaCl or (7) MgCl₂. Salt concentrations are 50 mM MgCl₂ or 150 mM NaCl. Lipid concentration is 20 mM with a 40: 20,000 quinone to lipid mole ratio. Arrows indicate the instance when ascorbate was injected.

Figure 3

R_ox dependence on quinone concentration. Samples contain 50 mM cacodylate (pH 7.4), 1.0 mM ascorbate and 20 mM DMPC LUVs with the specified quinone concentration. Dark symbols correspond to solutions containing 50 mM MgCl₂ and white symbols to solutions containing 150 mM NaCl.

Figure 4

EPR spectra of deareated solutions obtained after reduction of LQ in the presence or absence of MgCl₂ and in the presence or absence of 20 mM DMPC or DMPC/DMPA in 50 mM cacodylate, pH 7.4. Detailed composition of samples and relative amounts of semiquinone species are shown in Table II. Simulated and optimized spectra are shown with dotted lines. The optimization procedure is described in the “Experimental” section. EPR hyperfine splitting constants are those previously reported for the free and magnesium-complexed LQ^·− [7].

Figure 5

EPR spectra of deareated solutions obtained after reduction of PHQ and NQ in the presence or absence of MgCl₂ and in the presence or absence of 20 mM DMPC or DMPC/DMPA in 50 mM cacodylate, pH 7.4. Detailed composition of samples and relative amounts of semiquinone species are shown in Table II. Simulated spectra are shown with dotted lines. The optimization procedure is described in “Materials and methods”. EPR hyperfine splitting constants are those previously reported for the free and magnesium-complexed PHQ^·− and for the free ion of NQ^·− [7].