Direct Comparison of Electron Transfer Properties of Two Distinct Semisynthetic Triads with Non-Protein Based Triad: Unambiguous Experimental Evidences on Protein Matrix Effects Yi-Zhen Hu, [a] Hiroshi Takashima, [a] Shinya Tsukiji, [a] Seiji Shinkai, [a] Teruyuki Nagamune, [b] Shigero Oishi, [c] and Itaru Hamachi* [a] Abstract: In order to understand the roles of protein matrix in electron trans- fer processes (ET) within biological systems, a heme-based donor (Zn-heme: ZnPP)-sensitizer (Ru 2 (bpy) 3 )-acceptor (cyclic viologen: BXV 4 ) triad 1 was used as a probe molecule. Two semi- synthetic systems, Cyt-b 562 (1) and Mb(1), in which the triad is incorporated into cytochrome b 562 (Cyt-b 562 ) or into myoglobin (Mb), were constructed by cofactor reconstitution. These two semi- synthetic proteins were compared with the triad itself (i.e., without the protein matrix) using absorption spectroscopy, steady state emission and excitation studies, laser flash photolysis experi- ments, and molecular modeling. Photo- excitation of the ZnPP moiety of Cyt- b 562 (1) or Mb(1) leads to a direct ET from the triplet state of ZnPP state ( 3 ZnPP) to BXV 4 to generate a final charge-separated (CS) state, Cyt- b 562 (Zn  )-Ru 2 -BXV 3. or Mb(Zn  )- Ru 2 -BXV 3. . On the other hand, direct ET from the excited ZnPP moiety to the BXV 4 moiety is also involved in 1 in the absence of the protein matrix, but the excited state of ZnPP involved is not 3 ZnPP, but the singlet excited state ( 1 ZnPP) in this pathway. When the Ru 2 (bpy) 3 moiety of Cyt-b 562 (1) or Mb(1) is excited, a stepwise ET relay occurs with the ion-pair, Cyt-b 562 (Zn)- Ru 3 -BXV 3. or Mb(Zn)-Ru 3 -BXV 3 . , as an intermediate, leading to the same final CS state as that generated in the direct ET pathway. The lifetimes of the corresponding final CS states were de- termined to be 300 ns for 1 in the absence of the protein matrix, 600± 900 ns for Cyt-b 562 (1) and 1.1 ± 18 ms for Mb(1), the values of which are greatly affected by the protein matrix. Molec- ular modeling study of the three systems consistently explained the differences of their photophysical behavior. Keywords: artificial photosynthesis ´ long-lived charge separation ´ photoinduced electron transfer ´ protein matrix effect ´ semisynthetic system Introduction Effects of protein matrix on biological electron transfer pro- cesses (ET) have been actively discussed for more than one decade in the fields of chemistry and biology. [1] It is proposed that, in addition to fixed donor ± acceptor distances and orien- tations, [2] other factors, such as the specific structural features in proteins and the dipole moments produced by a specific conformation of polypeptide chains and a hydrogen-bonding network, [3] may control biological ET. Considerable efforts gave valuable insight not only into understanding ET in biological system, but also into designing protein-based photoelectric devices. [4] However, many key questions con- cerning biological ETremain, for example: How does ETrate depend on distance? How do proteins ensure biological spe- cificity in ET? Rational design and syntheses of semisynthetic systems and detailed comparison of the ET events in the presence and in the absence of the protein matrix should help to answer these questions. Nevertheless, such a comparison is rather difficult. Recently, we and other groups independently reported protein-based semisynthetic photoreaction centers as artificial photosynthetic models. [5] Covalently connected donor ± acceptor dyad or triad was successfully hybridized to a protein matrix in these systems, rendering it possible to compare ET events in a protein matrix with those in a homogeneous solvent. [a] Prof. I. Hamachi, [ =] Dr. Y.-Z. Hu, H. Takashima, S. Tsukiji, Prof. S. Shinkai Department of Chemistry and Biochemistry Graduate School of Engineering Kyushu University, Fukuoka 812-8581 (Japan) Fax: ( 81)92-642-3611 E-mail: itarutcm@mbox.nc.kyushu-u.ac.jp [ = ] Visiting professor of the Institute of Molecular Science at Okazaki (Japan) [b] Prof. T. Nagamune Department of Chemistry and Biotechnology Graduate School of Engineering, The University of Tokyo Hongo, Bunkyo-ku, 113-8656 (Japan) [c] Prof. S. Oishi Department of Chemistry, School of Science Kitasato University Sagamihara, Kanagawa 228-8520 (Japan) FULL PAPER Chem. Eur. J. 2000, 6, No. 11  WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000 0947-6539/00/0611-1907 $ 17.50+.50/0 1907