DESIGN, SYNTHESIS, AND CHARACTERIZATION OF A PHOTOACTIVATABLE FLAVOCYTOCHROME MOLECULAR MAQUETTE

We report the construction of a synthetic flavo-heme protein that inco rporates two major physiological activities of flavoproteins: light ac tivation of flavin analogous to DNA photolyase and rapid intramolecula r electron transfer between the flavin and heme cofactors as in severa l oxidoreductases. The functional tetra-alpha-helix protein comprises two 62-aa helix-loop-helix subunits. Each subunit contains a single cy steine to which flavin (7-acetyl-10-methylisoalloxazine) is covalently attached and two histidines appropriately positioned for bis-his coor dination of heme cofactors. Both flavins and hemes are situated within the hydrophobic core of the protein. Intramolecular electron transfer from flavosemiquinone generated by photoreduction from a sacrificial electron donor in solution was examined between protoporphyrin IX and 1-methyl-2-oxomesoheme XIII. Laser pulse-activated electron transfer f rom flavin to meso heme occurs on a 100-ns time scale, with a favorabl e free energy of approximately - 100 meV. Electron transfer from fIavi n to the lower potential protoporphyrin IX, with an unfavorable free e nergy, can be induced after a lag phase under continuous light illumin ation. Thus, the supporting peptide matrix provides an excellent frame work for the positioning of closely juxtaposed redox groups capable of facilitating intramolecular electron transfer and begins to clarify i n a simplified and malleable system the natural engineering of flavopr oteins.