FIGURE 4.

Difference FT-IR spectra showing the effect of solvent isotope exchange on the azide antisymmetric stretching band in chloride-depleted PSII samples. Difference spectra were created by ratio of data taken before and after flash excitation, followed by conversion to absorbance. In A and B, the results of three consecutive 532 nm laser flashes to a preflashed, dark adapted PSII sample are presented in red (flash 1), green (flash 2), and blue (flash 3). In A, chloride-depleted PSII in ¹H₂O p¹H 7.5 buffer contained 15 mM (average of 39). In B, chloride-depleted PSII in ²H₂O p²H 7.5 buffer contained 15 mM (average of 26). In C, an isotope-edited, ¹H₂O-minus-²H₂O, double difference spectrum was generated by subtracting (B, flash 3, blue) from (A, flash 3, blue). In D, a dark-minus-dark control was generated from the data set in A. In E, a control double difference spectrum, in which no vibrational bands are expected, was generated by subtraction of one half of the data in A from the other half of the data set and dividing by the square root of two. Delta absorbance on the y axis indicates that the spectra show changes in absorbance. All samples contained 1.5 mM recrystallized DCBQ. The tick marks on the y axis represent 5 × 10⁻⁴ AU.