Hemin-stimulated docking of cytochrome c to a hemin-DNA aptamer complex.

DNA aptamers were selected for their ability to bind simultaneously to the protein cytochrome c and to the metalloporphyrin hemin. Such aptamers each contained a conserved guanine-rich core, analogous to sequences shown previously to form a hemin-binding site when folded. The detailed study of CH6A, a deletion mutant of one clone, indicated that in the presence of hemin the guanine-rich core of the aptamer folded to form a guanine quadruplex. Both hemin and potassium ions were required for this folding. The binding of fully oxidized cytochrome c to this DNA-hemin complex resulted in an absorbance difference spectrum in the Soret region, which could be used as an indicator of binding behavior. It was found that cytochrome c bound more tightly to the folded CH6A DNA-hemin complex than to the folded CH6A DNA alone. A single hemin molecule and a single cytochrome c bound to each molecule of folded CH6A. Footprinting experiments showed the binding site of the cytochrome c to be a partial duplex element of the aptamer, immediately flanking its guanine-rich hemin-binding site. The order of addition of hemin and cytochrome c appeared not to affect either the formation rate or the structure of the final ternary complex. The ternary complex represents the docking of a nucleic acid-heme complex to cytochrome c (a protein-heme complex). Future experiments will focus on investigating the optimal electron-transfer path between the two iron centers through intervening protein and DNA.