Synthesis and Photophysical Properties of Zinc Myoglobin Appending an Ethidium Ion as a DNA Intercalator

Hiroshi Takashima, Yukiko Matsushima, Yasuyuki Araki, Osamu Ito, and Keiichi Tsukahara

J. Biol. Inorg. Chem., 13, 171-181 (2008).


In order to elucidate the intramolecular photoinduced electron-transfer or energy-transfer mechanism of the zinc myoglobin (ZnMb) dyad and to construct a photoreaction system within a Mb-DNA complex, we newly prepare ZnMb appending an ethidium ion (Et+).  The steady-state fluorescence of ZnMb-Et+ at 600 nm and its lifetime (2.2 ns) indicate that the excited singlet state of 1(ZnMb)* is not quenched by the Et+ moiety, whereas the lifetime of the excited triplet state of 3(ZnMb)*-Et+ was shorter (4.3 ms) than those of ZnMb and the intermolecular (ZnMb + ethidium) system.  Upon photoirradiation of Et+, fluorescence studies indicated the intramolecular quenching reactions from the excited singlet state, 1(Et+)*, to ZnMb, the process of which is likely the photoinduced energy-transfer reaction via a through-space mechanism.  We also demonstrate the photophysical and spectroscopic properties of ZnMb-Et+ in the presence of calf thymus (CT) DNA.  The changes in the absorption and fluorescence spectra of ZnMb-Et+ on the addition of CT-DNA up to 15 equiv were very small, indicating that there are no major changes in the heme pocket.  However, we observed a longer lifetime of 3(ZnMb)*-Et+ in the presence of CT-DNA (5.3 ms) by single flash photolysis.  This was induced by noncovalent interactions between Et+ and CT-DNA, followed by conformational change of Et+ at the surface of ZnMb, where the donor-acceptor distance was probably elongated by CT-DNA.  The synthetic manipulation at the Mb surface, by using a DNA-intercalator coupled with photoinduced reaction, may provide a sensitive transient signal for DNA and valuable information to construct new Mb-DNA complex.