No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

Abstract

In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn(4) cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S(0) --> S(1), S(1) --> S(2), and S(2) --> S(3) transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm(-)(1)) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 A from the Mn(4) cluster and accepts a hydrogen bond from Tyr Y(D)). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn(4) cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S(0) --> S(1), S(1) --> S(2), or S(2) --> S(3) transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn(4) cluster during the S(1) --> S(2) transition be localized on the Mn ion that is ligated by the alpha-COO(-) group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn(4) cluster during the S(0) --> S(1) and S(2) --> S(3) transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn(4) cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn(4) cluster that occurred during the collection of the X-ray diffraction data.

FIGURE 1

Comparison of the mid-frequency FTIR difference spectra of wild-type, D1-E189Q, and D1-E189R PSII particles in response to the first (A), second (B), third (C), and fourth (D) of six successive flash illuminations applied at 273 K [traces (a) in each panel]. The spectra (plotted from 1760 cm⁻¹ to 1170 cm⁻¹) correspond predominantly to S₂-minus-S₁, S₃-minus-S₂, S₀-minus-S₃, and S₁-minus-S₀ FTIR difference spectra, respectively. Spectra of two wild-type samples [WT(D1a) and WT(D1b)] are shown in each panel. The WT(D1a), D1-E189Q, and D1-E189R samples were exchanged into FTIR analysis buffer by passage through a centrifugal gel filtration column, while the WT(D1b) sample was exchanged by repeated concentration/dilution cycles (see text for details). To facilitate comparisons, the D1-E189Q and D1-E189R spectra have been multiplied by factors of ∼ 1.4 and ∼ 1.1, respectively, to maximize overlap with the wild-type spectra. Double-difference spectra (b, c, d in each panel) were calculated by directly subtracting the normalized traces shown in (a). The horizontal dashed lines indicate the zero levels. In (E), dark-minus-dark control traces of the WT(D1a), WT(D1b) , D1-E189Q, and D1-E189R PSII particles are included to show the noise levels. The color coding in (E) is the same as in (a) of panels A – D. Each spectrum represents the averages from nine samples (10,800 scans).

FIGURE 2

Comparison of the mid-frequency FTIR difference spectra of wild-type and D2-H189Q PSII particles in response to the first (A), second (B), third (C), and fourth (D) of six successive flash illuminations applied at 273 K [traces (a) in each panel]. The spectra (plotted from 1760 cm⁻¹ to 1170 cm⁻¹) correspond predominantly to S₂-minus-S₁, S₃-minus-S₂, S₀-minus-S₃, and S₁-minus-S₀ FTIR difference spectra, respectively. Spectra of two wild-type samples [WT(D1a) and WT(D2)] are shown in each panel. The mutant and both wild-type samples were exchanged into FTIR analysis buffer by passage through a centrifugal gel filtration column (see text for details). The WT(D1a) spectra are reproduced from Figure 1. To facilitate comparisons, the D2-H189Q spectrum was multiplied by a factor of ∼ 1.8 to maximize overlap with the wild-type spectra. Double-difference spectra (b, c in each panel) were calculated by directly subtracting the normalized traces shown in (a), except that the WT(D1a)-minus-WT(D1b) double-difference spectra in (c) are reproduced from Figure 1. The horizontal dashed lines indicate the zero levels. In (E), dark-minus-dark control traces of the WT(D1a), WT(D2) , and D2-H189Q PSII particles are included to show the noise levels. The color coding in (E) is the same as in (a) of panels A – D. Each wild-type spectrum represents the averages from nine samples (10,800 scans). The D1-H189Q spectrum represents the average from ten samples (12,000 scans).