Time-resolved FTIR difference spectroscopy in combination with specific isotope labeling for the study of A1, the secondary electron acceptor in photosystem 1

Abstract

A phylloquinone molecule (2-methyl, 3-phytyl, 1, 4-naphthoquinone) occupies the A(1) binding site in photosystem 1 particles from Synechocystis sp. 6803. In menB mutant photosystem 1 particles from the same species, plastoquinone-9 occupies the A(1) binding site. By incubation of menB mutant photosystem 1 particles in the presence of phylloquinone, it was shown in another study that phylloquinone will displace plastoquinone-9 in the A(1) binding site. We describe the reconstitution of unlabeled ((16)O) and (18)O-labeled phylloquinone back into the A(1) binding site in menB photosystem 1 particles. We then produce time-resolved A(1)(-)/A(1) Fourier transform infrared (FTIR) difference spectra for these menB photosystem 1 particles that contain unlabeled and (18)O-labeled phylloquinone. By specifically labeling only the phylloquinone oxygen atoms we are able to identify bands in A(1)(-)/A(1) FTIR difference spectra that are due to the carbonyl (C=O) modes of neutral and reduced phylloquinone. A positive band at 1494 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum is found to downshift 14 cm(-1) and decreases in intensity on (18)O labeling. Vibrational mode frequency calculations predict that an antisymmetric vibration of both C=O groups of the phylloquinone anion should display exactly this behavior. In addition, phylloquinone that has asymmetrically hydrogen bonded carbonyl groups is also predicted to display this behavior. The positive band at 1494 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum is therefore due to the antisymmetric vibration of both C=O groups of one electron reduced phylloquinone. Part of a negative band at 1654 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum downshifts 28 cm(-1) on (18)O labeling. Again, vibrational mode frequency calculations predict this behavior for a C=O mode of neutral phylloquinone. The negative band at 1654 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum is therefore due to a C=O mode of neutral phylloquinone. More specifically, calculations on a phylloquinone model molecule with the C(4)=O group hydrogen bonded predict that the 1654 cm(-1) band is due to the non hydrogen bonded C(1)=O mode of neutral phylloquinone.

FIGURE 1

A view of A_1-A and its environment. Possible H-bonds are shown as dashed lines. The B-side binding site is very similar. The carbonyl oxygen atoms of A_1-A are labeled 1 and 4. The various oxygen and nitrogen atoms of the protein backbone and amino acid side chains are also labeled. Figure generated using Swiss PDBViewer (35) from the crystallographic coordinates of PS1 at 2.5 Å resolution (9) (PDB file accession number 1JB0).

FIGURE 2

(A) FTIR absorption spectra for unlabeled (¹⁶O) (dotted) and ¹⁸O-labeled PhQ (solid) in THF. The spectra are scaled so that the intensity of the broad bands below 1500 cm⁻¹ are similar. The ratio of the intensity of the 1662 cm⁻¹ band in the two spectra is 0.30. Based on this assay we conclude that ∼70% of the PhQ carbonyl oxygen atoms are ¹⁸O-labeled. (B) ¹⁸O spectrum from A with 30% of the ¹⁶O spectrum subtracted from it. The resulting spectrum was then divided by 0.7 so that the bands below 1500 cm⁻¹ were again of the same intensity. The ratio of the intensities of the two bands at 1662 and 1634 cm⁻¹ indicate that ¹⁸O labeling leads to an ∼24% decrease in intensity of the C=O band of PhQ. (C) Calculated IR spectra obtained from DFT calculations using PhQ model shown in the inset. The absorbance scale does not apply to the calculated spectra in C. The calculated intensities are in km/mol and the 1661 cm⁻¹ mode has an intensity of 369 km/mol (30). The frequency axis for the spectra in (C) have been scaled by 0.965, which is normal for calculations using the B3LYP functional and the 6-31G+(d) basis (31).

FIGURE 3

Calculated IR spectra obtained from DFT calculations using PhQ model shown in the inset, with the C₄=O H-bonded to the NH group of the peptide backbone of a truncated leucine residue. As in Fig. 2, frequencies have been scaled by 0.965 cm⁻¹. Part of the IUPAC numbering scheme of PhQ is also shown in the inset. Intensity scale is km/mol.

FIGURE 4

(A) Calculated IR spectra for unlabeled (¹⁶O) (dotted) and ¹⁸O-labeled (solid) PhQ⁻. (B) Calculated IR spectra for unlabeled (¹⁶O) (dotted) and ¹⁸O-labeled PhQ⁻ (solid) in the presence of a truncated leucine residue. The one electron reduced form of the molecular models shown in the insets were used. Calculated frequencies have been scaled by a factor of 0.965. The calculated (¹⁸O–¹⁶O) double difference spectra for both molecular models (with or without H-bond) are also shown (middle). The central idea is that the band at 1480–1482 cm⁻¹ in the unlabeled spectra corresponds to the band at ∼1495 cm⁻¹ in the unlabeled experimental spectrum in Fig. 5 below. In addition, the band at 1466–1467 cm⁻¹ in the calculated ¹⁸O-labeled spectra corresponds to the band at ∼1480 cm⁻¹ in the ¹⁸O-labeled experimental spectrum in Fig. 5 (or Fig. 6).

FIGURE 5

FTIR DS obtained using menB mutant PS1 particles reconstituted with (A) unlabeled (¹⁶O) (solid) and (B) ¹⁸O-labeled (dotted) PhQ. (C) (¹⁸O–¹⁶O) FTIR double difference spectrum. (D) Time-resolved spectrum collected before the laser flash (for the sample containing ¹⁸O-labeled PhQ). Spectrum D is the average of 9 spectra collected in 5 μs increments before the laser flash. It was collected in a manner identical to that described previously (11), and gives a measure of the noise level in the experiment. The spectra in A/B are the average of three/two measurements on different samples, respectively. Spectrum E/F shows the SD of the three/two spectra used to obtain spectra A/B, respectively. These SD spectra give a true measure of the noise in the FTIR DS.

FIGURE 6

Same spectra as in Fig. 5 but on an expanded scale in the 1505–1470 cm⁻¹ region. (Top) Unlabeled (solid line) and ¹⁸O-labeled (dotted line) FTIR DS. (Middle) The three measures of the noise level (D–F). (Bottom) (¹⁸O–¹⁶O) FTIR double difference spectrum. The length of the four thick vertical bars represents absorption difference amplitudes. Clearly, the amplitude of the bands at 1494 and 1480 cm⁻¹ in the ¹⁶O and ¹⁸O-labeled spectra, respectively, are at least a factor of two above all three measures of the noise level, and the derivative feature in the double difference spectrum is three to four times above the noise level.