Reaction of Oxidized CuZnSOD with Polyphenols

Abstract

Several polyphenolic compounds, including twelve flavonols and a variety of lower molecular weight compounds were found to diminish the effects of CuZnSOD on NMR relaxation of fluoride ion in a pH and concentration dependent manner. While we originally thought the effect arose from binding of the compounds to the enzyme active site, several lines of evidence indicated that active polyphenols reduced the paramagnetic copper(II) form of the enzyme to the diamagnetic copper(I) form, thereby giving false positive indications of enzyme inhibition in the NMR assay. First, docking experiments failed to provide a satisfactory explanation of the SAR. Second, effects on the enzyme’s EPR spectrum indicated that catechols could bind the active site copper leading to enzyme reduction. Third, while these reactions did not proceed to completion in aerobic solution, they did so under inert atmosphere. Fourth, experiments employing superoxide producing compounds demonstrated that loss of NMR activity did not prevent the enzyme from redox cycling. Thus, while the polyphenols appeared to inhibit the enzyme in the NMR assay, the compounds did not inhibit the enzyme’s reactions with superoxide.