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.
Chinnapen
Daniel J F
DJ
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada.
Sen
Dipankar
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D054856:G-Quadruplexes; D006427:Hemin / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017384:Sequence Deletion; D013056:Spectrophotometry, Ultraviolet; D013463:Sulfuric Acid Esters / Q000737:chemistry
D003574:Cytochrome c Group; D009841:Oligonucleotides; D013463:Sulfuric Acid Esters; D006427:Hemin; D004247:DNA; C007482:dimethyl sulfate; D004047:Diethyl Pyrocarbonate