2'-FLUORO-2'-DEOXY-D-ARABINOFLAVIN - CHARACTERIZATION OF A NOVEL FLAVIN AND ITS EFFECTS ON THE FORMATION AND STABILITY OF 2-ELECTRON-REDUCED MERCURIC ION REDUCTASE

With the goal of generating a novel fluorine-containing flavin analogu e with a reduction potential the same as normal flavin, 2'-fluoro-2'-d eoxy-D-arabinoflavin has been synthesized. In its riboflavin and FAD f orms, UV-visible spectral properties are similar to those of normal fl avins, and tight binding to riboflavin binding protein and mercuric io n reductase occurs with very similar spectral changes. The reduction p otential of the 2'-FaFAD analogue is determined to be -207 mV compared with -206 mV for FAD, indicating that the intervening 1'-methylene gr oup insulates the redox-active isoalloxazine from the 2'-fluorine. Wit h the intent of using the analogue as a fluorine NMR probe of the acti ve site environments of two-electron-reduced mercuric ion reductase, a poenzyme was reconstituted and its behavior under reducing conditions examined. Whereas with normal enzyme, addition of two electrons gives rapid formation of a charge-transfer species where FAD remains oxidize d and a disulfide is reduced to a thiol/ thiolate pair, with the 2'-Fa FAD enzyme, addition of two electrons gives rapid reduction of the fla vin followed by slow transfer of electrons to the disulfide with very little development of the typical charge-transfer absorption. Analysis of crystal structure data suggests that having the fluorine in the al ternate arabino stereochemistry places it much nearer the flavin-proxi mal cysteine/cystine sulfur, where it may inhibit both electron transf er from reduced flavin and the charge-transfer interaction between red uced thiolate and FAD.