Time-resolved FTIR difference spectroscopy for the study of photosystem I particles with plastoquinone-9 occupying the A1 binding site

In photosystem I from plants and cyanobacteria a phylloquinone molecule, called A1, functions as the secondary electron acceptor. In cyanobacteria, genes that encode for proteins involved in phylloquinone biosynthesis can be deleted. Here, we have studied three different gene deletion mutants called menB, menD, and menE mutants. In these mutants, plastoquinone-9 occupies the A1 binding site. Using time-resolved, step-scan FTIR difference spectroscopy we have produced A1 - /A1 FTIR difference spectra for menB, menD, and menE photosystem I particles at 77 K. These difference spectra show that the P700 triplet state ( 3 P700) is formed in a large fraction of the particles. Infrared spectral signatures that are not due to 3 P700 are also observed in the spectra and are suggested to be associated with plastoquinone-9 anion formation in a portion of the particles. By subtracting the known 3 P700 spectral signatures, we produce an A1 - /A1 FTIR difference spectrum for PS I particles with plastoquinone-9 occupying the binding site. This spectrum shows that a band that we have previously assigned to a C-O mode of the phylloquinone anion in WT A1 - /A1 FTIR DS down-shifts 8c m -1 when plastoquinone-9 occupies the A1 binding site. Using density functional theory type calculations to produce anion minus neutral infrared difference spectra for both phylloquinone and plastoquinone-9, it is shown that such a downshift is reasonable. A1 - /A1 FTIR difference spectra, obtained using menB mutant photosystem I particles that were incubated in the presence of phylloquinone, are found to be very similar to those obtained using normal WT photosystem I particles. This result indicates that we were able to reincorporate phylloquinone back into the A1 binding site and that the reincorporated phylloquinone and its immediate protein environment, in both the neutral and anion state, are very similar to that found in wild type photosystem I particles. For the reconstituted menB mutant photosystem I particles, no spectral signatures associated with 3 P700 are observed, indicating that phylloquinone occupies the A1 site in all of the reconstituted