2'-fluoro-2'-deoxy-D-arabinoflavin: characterization of a novel flavin and its effects on the formation and stability of two-electron-reduced mercuric ion reductase.

Abstract

With the goal of generating a novel fluorine-containing flavin analogue with a reduction potential the same as normal flavin, 2'-fluoro-2'-deoxy-D-arabinoflavin has been synthesized. In its riboflavin and FAD forms, UV-visible spectral properties are similar to those of normal flavins, and tight binding to riboflavin binding protein and mercuric ion reductase occurs with very similar spectral changes. The reduction potential of the 2'-FaFAD analogue is determined to be -207 mV compared with -206 mV for FAD, indicating that the intervening 1'-methylene group insulates the redox-active isoalloxazine from the 2'-fluorine. With the intent of using the analogue as a fluorine NMR probe of the active site environments of two-electron-reduced mercuric ion reductase, apoenzyme 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 oxidized and a disulfide is reduced to a thiol/thiolate pair, with the 2'-FaFAD enzyme, addition of two electrons gives rapid reduction of the flavin 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 alternate arabino stereochemistry places it much nearer the flavin-proximal cysteine/cystine sulfur, where it may inhibit both electron transfer from reduced flavin and the charge-transfer interaction between reduced thiolate and FAD.