DNA and RNA Catalysts with Peroxidase Activity: An Investigation into Structure and Mechanism

A new category of catalytic nucleic acids has been characterized. A specific single-stranded DNA and its corresponding RNA version (“PS2.M” and “rPS2.M,” respectively) define a new kind of cofactor-utilizing catalytic nucleic acid, in which the nucleic acid moiety utilizes the porphyrin hemin to catalyze oxidative chemistries.
The PS2.M and rPS2.M “aptamers” (from the latin “aptus” =to fit) were shown to complex tightly to hemin and found to exhibit peroxidative (oxidation of organic/inorganic substrates by hydrogen peroxide) activity two orders of magnitude higher than that of uncomplexed monomeric hemin (or hemin in the presence of control DNA oligomers). DNA and RNA-catalyzed peroxidation reactions were contingent on the binding of hemin to specific folded structures for the catalytic oligomers (G-quadruplex structures, stabilized by potassium ions). Optical and EPR (Electron Paramagnetic Resonance) studies indicated that changes in the coordination of the hemin iron complex occurred upon binding to the
aptamer, such that these complexes more closely resembled the horseradish peroxidase protein enzyme than uncomplexed hemin.
An extensive kinetic analysis of the PS2.M-hemin- and rPS2.M-hemin-catalyzed peroxidations provided valuable insights into the reaction mechanism. Catalysis by the DNA/RNA enzymes proved to occur through an accelerated formation of the “Compound I” intermediate following the breakdown of a hemin-hydrogen peroxide covalent complex.
Spectroscopic data suggested that the Compound I species was a ferryl (FeIV) activated porphyrin p-cation radical, which carried a two-electron oxidizing equivalent.
A role for general buffer catalysis by nitrogenous buffers was also clearly established, with both the basic and the acidic components contributing to the increase of peroxidation rates. Investigation of the “alkaline” transition for the aptamer-hemin complexes indicated that the DNA/RNA apoenzymes provided a polar environment and possibly hydrogen bond(s) at the axial positions of the hemin (with hydrophobic interactions around the porphyrin ring). Analysis of the pH dependence of the catalyzed peroxidation rates showed that the high-spin aquo-hemin species and the basic component of the buffer were the “active species” responsible for the maximal DNA/RNA enzyme activity. Finally, EPR spectroscopy and chemical-probing studies identified specific guanine bases in the PS2.M sequence, which specifically interacted (likely via coordination) towards
activating the bound hemin for peroxidations. The above experimental information was used to formulate a model for the folding of the PS2.M DNA, and to construct a model for structure-function relationships within this enzyme.