Interactions between heme and G-quadruplex DNA involve the oxygen of guanine

Guanine quadruplexes are non-canonical DNA structures with various functions including transcription and translation regulation and telomere protection. These structures are known to bind the heme prosthetic group, resulting in heme-bound G-quadruplexes (heme-G4) that exhibit enhanced peroxidase activity and act as promising biocatalysts. The structure of the heme-G4, specifically how the DNA scaffold interacts with the heme iron, is key in understanding the catalytic mechanism of these DNAzymes. In heme proteins, the nature of a heme axial ligand plays an essential role in manipulating the inherent reactivity of the heme prosthetic group. Several proposals regarding the heme axial ligand in the heme-G4 complexes have been previously presented, including nitrogen or oxygen atoms of the guanine base or a water molecule sandwiched between the heme macrocycle and the DNA quadruplex. Despite numerous studies, no convincing experimental evidence has yet been provided as to the nature of the key proximal ligand. In this work, we present extensive electronic absorption and resonance Raman spectroscopic studies of ferric and ferrous heme-G4 complexes, including their ligated forms. Our studies provide experimental evidence that the oxygen atom of the guanine base acts as an axial ligand supported by detection of the ν(Fe-O_G) stretching mode at 563 cm^-1 in the spectra of ferric heme-G4. These results provide structural data that can help understand the mechanistic principles behind the observed enhanced peroxidase activity of heme-G4 quadruplexes and aid in design of advanced biocatalysts.