The A_-1 pigment in photosystem I contributes to (P₇₀₀⁺-P₇₀₀) FTIR difference spectra

Previously, photo-accumulated (P700 +-P700) FTIR difference spectra have been obtained for many types of native and mutant PSI samples, and for PSI samples that have been globally and specifically isotope labeled. In nearly all cases interpretation of bands in to (P700 +-P700) FTIR difference spectra is based on the notion that P700 is a dimeric chlorophyll a species in both the neutral and cation radical forms. This widely held interpretation, that only two chlorophyll pigments contribute to (P700 +-P700) FTIR difference spectra, is called into question here on the basis of FTIR difference spectra that have been collected for mutant PSI samples in which the ligand to the A-1 pigments on the A and B branches have been modified. Photo-accumulated (P700 +-P700) FTIR difference spectra have been obtained for native PSI samples, and for mutant PSI samples where asparagine (N) at PsaA-601 and PsaB-587 were changed to lysine (K) or aspartic acid (D). Asparagine at A601/B587 is H-bonded to a water molecule that in turn ligates the A-1B/A-1A accessory chlorophyll a pigment, respectively. The NK or ND mutations induce large changes in the (P700 +-P700) FTIR difference spectra indicating that these spectra cannot simply be interpreted in terms of only the vibrational modes of the PA and PB pigments that are normally considered to comprise P700. Mutation induced band-shifts are most effectively visualized via “mutant minus wildtype” double difference spectra, and here we assign spectral features in these double difference spectra to the A-1A and A-1B pigments.