Evidence from FTIR difference spectroscopy of an extensive network of hydrogen bonds near the oxygen-evolving Mn(4)Ca cluster of photosystem II involving D1-Glu65, D2-Glu312, and D1-Glu329

Abstract

Analyses of the refined X-ray crystallographic structures of photosystem II (PSII) at 2.9-3.5 A have revealed the presence of possible channels for the removal of protons from the catalytic Mn(4)Ca cluster during the water-splitting reaction. As an initial attempt to verify these channels experimentally, the presence of a network of hydrogen bonds near the Mn(4)Ca cluster was probed with FTIR difference spectroscopy in a spectral region sensitive to the protonation states of carboxylate residues and, in particular, with a negative band at 1747 cm(-1) that is often observed in the S(2)-minus-S(1) FTIR difference spectrum of PSII from the cyanobacterium Synechocystis sp. PCC 6803. On the basis of its 4 cm(-1) downshift in D(2)O, this band was assigned to the carbonyl stretching vibration (C horizontal lineO) of a protonated carboxylate group whose pK(a) decreases during the S(1) to S(2) transition. The positive charge that forms on the Mn(4)Ca cluster during the S(1) to S(2) transition presumably causes structural perturbations that are transmitted to this carboxylate group via electrostatic interactions and/or an extended network of hydrogen bonds. In an attempt to identify the carboxylate group that gives rise to this band, the FTIR difference spectra of PSII core complexes from the mutants D1-Asp61Ala, D1-Glu65Ala, D1-Glu329Gln, and D2-Glu312Ala were examined. In the X-ray crystallographic models, these are the closest carboxylate residues to the Mn(4)Ca cluster that do not ligate Mn or Ca and all are highly conserved. The 1747 cm(-1) band is present in the S(2)-minus-S(1) FTIR difference spectrum of D1-Asp61Ala but absent from the corresponding spectra of D1-Glu65Ala, D2-Glu312Ala, and D1-Glu329Gln. The band is also sharply diminished in magnitude in the wild type when samples are maintained at a relative humidity of </=85%. It is proposed that D1-Glu65, D2-Glu312, and D1-Glu329 participate in a common network of hydrogen bonds that includes water molecules and the carboxylate group that gives rise to the 1747 cm(-1) band. It is further proposed that the mutation of any of these three residues, or partial dehydration caused by maintaining samples at a relative humidity of <or=85%, disrupts the network sufficiently that the structural perturbations associated with the S(1) to S(2) transition are no longer transmitted to the carboxylate group that gives rise to the 1747 cm(-1) band. Because D1-Glu329 is located approximately 20 A from D1-Glu65 and D2-Glu312, the postulated network of hydrogen bonds must extend for at least 20 A across the lumenal face of the Mn(4)Ca cluster. The D1-Asp61Ala, D1-Glu65Ala, and D2-Glu312Ala mutations also appear to substantially decrease the fraction of PSII reaction centers that undergo the S(3) to S(0) transition in response to a saturating flash. This behavior is consistent with D1-Asp61, D1-Glu65, and D2-Glu312 participating in a dominant proton egress channel that links the Mn(4)Ca cluster with the thylakoid lumen.

FIGURE 1

Comparison of the mid-frequency S₂-minus-S₁ FTIR difference spectra of wild-type PSII core complexes (A) maintained at a relative humidity of 99% (black) or 85% (blue) or as a dry film in the sample cell (red) or (B) exchanged into FTIR buffer containing H₂O (black) or D₂O (red) and maintained at a relative humidity of 99% (in an atmosphere of H₂O or D₂O, respectively). In (A), the spectra have been normalized to the peak-to-peak amplitudes of the negative ferricyanide peak at 2115 cm⁻¹ and the positive ferricyanide peak at 2038 cm⁻¹. In (B), the spectra have been normalized to maximize overlap between 1450 and 1350 cm⁻¹. The black, blue, and red traces in (A) represent the averages of four, seven, and four samples, respectively, and consist of 13,800, 24,200, and 13,400 scans, respectively. The black and red traces in (B) each represent the average of four samples and consist of 13,800 and 13,600 scans, respectively. The sample temperature was 273 K.

FIGURE 2

Comparison of the mid-frequency FTIR difference spectra of wild-type (black) and D1-D61A (red) PSII core complexes in response four successive flash illuminations applied at 273 K. The wild-type spectra correspond predominantly to the S₂-minus-S₁, S₃-minus-S₂, S₀-minus-S₃, and S₁-minus-S₀ FTIR difference spectra, respectively. The data (plotted from 1770 cm⁻¹ to 1170 cm⁻¹) represent the averages of nine wild-type and eight D61A samples (10,800 and 9,600 scans, respectively). To facilitate comparisons, the mutant spectra have been multiplied by factors of ~ 1.1 after normalization to the peak-to-peak amplitudes of the negative ferricyanide peak at 2115 cm⁻¹ and the positive ferricyanide peak at 2038 cm⁻¹ to maximize overlap with the wild-type spectra. Dark-minus-dark control traces are included to show the noise level (lower traces).

FIGURE 3

Comparison of the mid-frequency FTIR difference spectra of wild-type (black) and D1-E65A (red) PSII core complexes in response to four successive flash illuminations applied at 273 K. The data (plotted from 1770 cm⁻¹ to 1170 cm⁻¹) represent the averages of nine wild-type and twelve D1-D61A samples (10,800 and 14,400 scans, respectively). To facilitate comparisons, the mutant spectra have been multiplied by factors of ~ 0.83 after normalization to the peak-to-peak amplitudes of the negative ferricyanide peak at 2115 cm⁻¹ and the positive ferricyanide peak at 2038 cm⁻¹ to maximize overlap with the wild-type spectra. Dark-minus-dark control traces are included to show the noise level (lower traces).

FIGURE 4

Comparison of the mid-frequency FTIR difference spectra of Mn-depleted wild-type (black) and intact D2-E312A (red) PSII core complexes in response to the first of six successive flash illuminations applied at 273 K. The data (plotted from 1770 cm⁻¹ to 1170 cm⁻¹) represent the averages of nine Mn-depleted wild-type and nine D2-E312A samples (10,800 scans each). The fraction of D2-E312A PSII reaction centers lacking Mn₄Ca clusters (0.31) was estimated from the amplitude negative peak at 1706 cm⁻¹ (see text for details). Accordingly, the spectrum of the Mn-depleted wild-type sample shown in this figure was multiplied by a factor of 0.31 after normalization to the peak-to-peak amplitudes of the negative ferricyanide peak at 2115 cm⁻¹ and the positive ferricyanide peak at 2038 cm⁻¹.

FIGURE 5

Comparison of the mid-frequency FTIR difference spectra of wild-type (black) and D2-E312A (red) PSII core complexes in response to four successive flash illuminations applied at 273 K. The data (plotted from 1770 cm⁻¹ to 1170 cm⁻¹) represent the averages of nine wild-type and nine D2-E312A samples (10,800 scans each). The S₂-minus-S₁ FTIR difference spectrum of D2-E312A was corrected for the presence of a significant population of Mn-depleted PSII reaction centers (see text for details). To facilitate comparisons, the mutant spectra have been multiplied by factors of ~ 1.4 after normalization to the peak-to-peak amplitudes of the negative ferricyanide peak at 2115 cm⁻¹ and the positive ferricyanide peak at 2038 cm⁻¹ to maximize overlap with the wild-type spectra. Dark-minus-dark control traces are included to show the noise level (lower traces).

FIGURE 6

Comparison of the mid-frequency FTIR difference spectra of wild-type (black) and D1-E329Q (red) PSII core complexes in response to four successive flash illuminations applied at 273 K. The data (plotted from 1770 cm⁻¹ to 1170 cm⁻¹) represent the averages of nine wild-type and four D1-D329Q samples (10,800 and 4,800 scans, respectively). The spectra have been normalized to the peak-to-peak amplitudes of the negative ferricyanide peak at 2115 cm⁻¹ and the positive ferricyanide peak at 2038 cm⁻¹. Dark-minus-dark control traces are included to show the noise level (lower traces).

FIGURE 7

The Mn₄Ca cluster and its environment as depicted in the 2.9 Å crystallographic structural model of PSII from Thermosynechococcus elongatus (3BZ1) (5). The four residues discussed in this panel are depicted in bright yellow (Asp61, Glu65, and Glu329 of the D1 polypeptide) or bright orange (Glu312 of the D2 polypeptide). The Mn, Ca, and Cl ions are depicted as red, orange, and green spheres, respectively. Tyrosine Y_Z and the protein ligands of the Mn₄Ca cluster are depicted in light yellow or orange (residues of D1 and CP43, respectively). Portions of water access, proton egress, and O₂ egress channels identified in the 2.9 Å structural model are depicted in blue, gray, and maroon, respectively (the channel coordinates from ref. (35) were graciously provided by A. Zouni). In this model, the shortest distances between the carboxylate group and the nearest Mn ion is 4.6 Å, 10.8 Å, 11.3 Å, and 7.5 Å for D1-Asp61, D1-Glu65, D2-Glu312, and D1-Glu329, respectively (5).

FIGURE 8

The ν(C=O) region of the S₂-minus-S₁ FTIR difference spectra of (A) wild-type PSII core complexes maintained at a relative humidity of 99% (black) or 85% (blue) or as a dry film in the sample cell (red), (B) wild-type PSII core complexes exchanged into FTIR buffer containing H₂O (black) or D₂O (red), (C) wild-type (black) and D1-D61A (red) PSII core complexes, (D) wild-type (black) and D1-E65A (red) PSII core complexes, (E) wild-type (black) and D1-E312A (red) PSII core complexes (after correction of D2-E312A for the presence of Mn-depleted reaction centers – see text for details), and (F) wild-type (black) and D1-E329Q (red) PSII core complexes. The data have been reproduced from Figures 1A, 1B, 2, 3, 5, and 6, respectively.