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 ( 16O) and 18O-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 18O-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 18O 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 18O 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.