KINETICS OF THE REDUCTIVE HALF-REACTION OF THE IRON-SULFUR FLAVOENZYME P-6-DEOXY-L-THREO-D-GLYCERO-4-HEXULOSE-3-DEHYDRASE REDUCTASE

The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideo xy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysacchar ide from Yersinia pseudotuberculosis V, This transformation is catalyz ed by two enzymes: P-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E(1)), which contains a pyridoxamine and a [2Fe-2S] center, and an NA DH-dependent P-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reduct ase (E(3)), which contains both an FAD and a [2Fe-2S] center. E(1) rea cts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and cataly zes the elimination of the hydroxyl at position 3 of the glucose moiet y, resulting in the formation of a covalently bound CDP-6-deoxy-Delta( 3,4)-glucoseen intermediate. E(3) transfers electrons from NADH to E(1 ), which uses these to reduce the Delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E(3) using both single wavelength and d iode array stopped flow absorbance spectroscopy. We find that NADH bin ds to both oxidized (K-d = 52.5 +/- 2 mu M) and two-electron-reduced ( K-d = 12.1 +/- 1 mu M) forms Of E(3), Hydride transfer from NADH to th e FAD moiety occurs at 107.5 +/- 3 s(-1) and exhibits a 10-fold deuter ium isotope effect when (4R)-[H-2]NADH is substituted for NADH, Follow ing the hydride transfer reaction, NAD(+) is released at 42.5 +/- 1 s( -1) and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer rea ction is pH-dependent with a pK(a) of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E (3). Finally, E(3) is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH. The [2Fe-2S] c enter of Eg was selectively disassembled by titration with mersalyl to give E(3)(apoFeS), The properties of this form of the enzyme are comp ared to those of the holoenzyme. Similarities and differences of the r eductive half-reactions of E(3) and related iron-sulfur flavoenzymes a re discussed.