FIGURE 7.

The kinetic isotope effects of the reduced enzyme specifically labeled with deuterium at the flavin N5 position in H₂O buffer. A, oxidation of the reduced enzyme labeled with N5-D was investigated using double-mixing stopped-flow spectrophotometry. During the first mixing, a solution of the oxidized enzyme (78 μm before mixing) was mixed with an equal volume of 100 μm 2-d-d-glucose (before mixing) in H₂O buffer under anaerobic conditions. The mixture was continued for 80 s after the first mixing, before the reduced enzyme was mixed with 100 mm sodium phosphate buffer containing 0.96 mm oxygen (final concentration) (filled circle line). Only H₂O buffers were employed in this experiment. The reaction was monitored at 395 nm to determine the rate constant for the H₂O₂ elimination from C4a-hydroperoxyflavin. The control experiment, where a solution of 100 μm d-glucose was used instead of 100 μm 2-d-d-glucose, was carried out in a similar fashion and is shown as a solid line trace. The results show that the kinetics of the trace with filled circles (N5-D reduced flavin) was similar to that of the reduced enzyme in D₂O buffer reacting with 0.96 mm oxygen in D₂O buffer (data in Fig. 2), whereas the kinetics of the double-mixing reaction employing d-glucose (solid line) was similar to the reaction carried out in H₂O buffer (dotted line trace, data from Fig. 2). The dotted line trace was multiplied by a factor of 0.7 and offset by + 0.005 absorbance units to bring the absorbance signal to a similar range as the traces from the double-mixing experiment. B, oxidation of the reduced enzyme labeled with N5-D was carried out using a single-mixing stopped-flow spectrophotometer. Equal volumes of solutions of the oxidized enzyme 88 μm in H₂O buffer (before mixing) in syringe A and 100 μm 2-d-d-glucose in H₂O buffer (before mixing) in syringe B were manually pushed and mixed in syringe D under anaerobic conditions. The reaction in syringe D was allowed to proceed for 80–100 s to obtain the completely reduced P2O labeled with N5-D before the single-mixing stopped-flow experiment took place by mixing syringe D solution with an aerobic buffer in syringe C. The final reaction contained 22 μm of the reduced enzyme specifically labeled with N5-D and 0.96 mm oxygen in 100 mm sodium phosphate in H₂O, pH 7.0 (open circle trace). The open circle trace is similar to the kinetic trace from the reduced enzyme in D₂O buffer reacting with 0.96 mm oxygen in D₂O buffer (Fig. 2). For a reference, the trace from Fig. 2B was multiplied by a factor of 0.66, offset by + 0.044 absorbance units and is shown as a dotted line. The solid line trace was obtained from the control reaction, in which a solution of 100 μm d-glucose was used instead of 100 μm 2-d-d-glucose, and the reaction was carried out in a similar fashion to that for the open circle trace. The results of the single-mixing stopped-flow in Fig. 7B are similar to the results of the double-mixing stopped-flow experiment in Fig. 7A. The data in this figure indicate that the solvent kinetic isotope effect on the H₂O₂ elimination from C4a-hydroperoxyflavin is mainly contributed by the N5-H bond breaking.