Electron Transfer Quenching and Photoinduced EPR of Hypericin and the Ciliate Photoreceptor Stentorin † Todd A. Wells, ‡ Aba Losi, § Renke Dai, ‡,| Paul Scott, ⊥ Su-Moon Park, # John Golbeck, ⊥ and Pill-Soon Song* ,‡ Departments of Chemistry and Biochemistry, UniVersity of Nebraska, Lincoln, Nebraska 68588-0304 ReceiVed: May 3, 1996; In Final Form: June 25, 1996 X Time-correlated single photon counting was used to observe dynamic quenching of the hypericin and stentorin excited singlet states. The fluorescence quenching data for hypericin and stentorin were interpreted in terms of electron transfer. The observed correlation between free energy change of electron transfer and quenching rate constant suggests that quenching proceeds via electron transfer from hypericin and stentorin to the quenchers. EPR spectra for hypericin, stentorin, and stentorin chromoprotein demonstrated that free radical formation was initiated or enhanced by visible light and that similar radical species were produced in each sample. Furthermore, the EPR signal for stentorin was significantly enhanced by 1,4-benzoquinone, but the overall shape and g-value was unchanged. We suggest that electron transfer in the excited state of these chromophores results in the formation of a cation radical. This electron transfer is a rapid and efficient pathway for deactivation of hypericin and stentorin excited singlet states and should be considered when discussing the photoreactivity of hypericin as a photodynamic agent and of stentorin as the Stentor coeruleus photoreceptor. Introduction Hypericin is a pigment of the naphthodianthrone family (Figure 1), found in plants of the genus Hypericum. 1 The ability of this quinoid molecule to produce hypericism, a condition of severe sensitivity to light, has been known for some time. 2,3 The photosensory ciliates, Stentor coeruleus and Blepharisma japonicum, use the hypericin-derived pigments stentorin and blepharismin, respectively, as the photoreceptor chromophores. 2 Recently, this photosensitizing pigment has been studied for its multitude of pharmacological activities and has been employed as an antidepressive agent, as an antitumoral agent, and as an antiviral agent. 4 Probably the most notable of these activities is hypericin’s ability to destroy the virus that causes equine infectious anemia 5 and its relative, the human immuno- deficiency virus, HIV. 6 The exact mechanism of hypericin’s therapeutic activity is not clear but the role of light initiation or enhancement has been established. Hypericin has been shown to produce singlet oxygen, 2,7,8 and much of the experimental data points to 1 O 2 as the source of the photodynamic activity. Superoxide (O 2 •- ) has been detected in DMSO solutions of hypericin, in aqueous suspensions of the hypericin lysine salt, 8,9 and in hypericin bound to artificial membranes. 10 Thus hypericin’s photobiological activity may also be superoxide radical mediated. Both type I and II quenchers suppressed photokilling of a human fibroblast cell line. 10,11 However, studies of ciliate photoreceptors, stentorins and blepharismins, and their primary photoprocesses have raised some doubt about the role of singlet oxygen and superoxide in hypericin-induced cellular and viral death. The ciliated protozoa S. coeruleus and B. japonicum contain hypericin-like pigments 3,12-17 sequestered in subpellicular gran- ules. 18,19 The recently elucidated structure of the stentorin chromophore 13 is given in Figure 1. Action spectra indicate that stentorin and blepharismin are responsible for the photo- induced motile responses of Stentor 20 and Blepharisma, 21 respectively. Although photokilling of both Stentor and Ble- pharisma under high light fluence is related to the formation of singlet oxygen, 7,22,23 it is unlikely that 1 O 2 initiates the signal cascade resulting in their photophobic and phototactic responses. On the other hand, the primary photoprocesses may originate from proton transfer. Indirect evidence for a light-driven pH decrease across the cell membrane has been found for Sten- tor, 14,20,24,25 and a light-induced acidification has recently been observed for hypericin inserted in phosphatidyl vesicles. 26 An intramolecular proton transfer to an appropriate amino acid has been proposed for the stentorin protein to explain an ultrafast bleaching process observed at 565-630 nm, 27 and proton transfer can be efficiently coupled to electron transfer. 28 † Dedicated to Professor Saburo Nagakura on his 75th birthday. * To whom correspondence should be addressed. Phone: (402) 472 2749. FAX: (402) 472 2044. E-mail: pssong@unl.edu. § Department of Physics, University of Parma, Viale dell Scienze, 43100 Parma, Italy. | Present address: Laboratory of Molecular Carcinogenesis, National Cancer Institute, NIH, Bethesda, MD 20892. ⊥ Department of Biochemistry, University of Nebraska. # Department of Chemistry, University of New Mexico, Albuquerque, NM 87131. X Abstract published in AdVance ACS Abstracts, January 1, 1997. Figure 1. Structures of hypericin and stentorin. 366 J. Phys. Chem. A 1997, 101, 366-372 S1089-5639(96)01258-3 CCC: $14.00 © 1997 American Chemical Society