FOURIER-TRANSFORM INFRARED DIFFERENCE SPECTROSCOPY SHOWS NO EVIDENCE FOR AN ENOLIZATION OF CHLOROPHYLL-A UPON CATION FORMATION EITHER INVITRO OR DURING P700 PHOTOOXIDATION

Molecular changes associated with the photooxidation of the primary electron donor P700 in photosystem I from cyanobacteria have been investigated with Fourier transform infrared (FTIR) difference spectroscopy. Highly resolved signals are observed in the carbonyl stretching frequency region of the light-induced FTIR spectra. In order to assign and to interpret these signals, the FTIR spectra of isolated chlorophyll a and pyrochlorophyll a (lacking the 10a-ester carbonyl) in both their neutral and cation states were investigated. Comparison of the redox-induced FTIR difference spectra of these two model compounds demonstrates that upon chlorophyll a cation formation in tetrahydrofuran the 7c-ester carbonyl is essentially unperturbed while the 10a-ester carbonyl is upshifted from 1738 to 1751 cm−1. For the 9-keto group, the shift is from 1693 to 1718 cm−1 in chlorophyll a and from 1686 to 1712 cm−1 in pyrochlorophyll a. The 1718-cm−1 band in the difference spectrum of chlorophyll a is thus unambiguously assigned to the 9-keto carbonyl of the cation. Comparison of the light-induced FTIR difference spectrum associated with the photooxidation of P700 in vivo with the difference FTIR spectrum of chlorophyll a cation formation leads to the assignment of the frequencies of the 9-keto carbonyl group(s) at 1700 cm−1 in P700 and at 1717 cm−1 in P700+. These assignments are taken to indicate free keto carbonyl(s) in both P700 and P700+ with no evidence for the participation either in chlorophyll a+ or in P700+ of the chlorophyll enol form previously suggested by Wasielewski et al. [Wasielewski, M. R., Norris, J. R., Shipman, L. L., Lin, C.-P., & Svec, W. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2957–2961] and more recently by Heald et al. [Heald, R. L., Callahan, P. M., & Cotton, T. M. (1988) J. Phys. Chem. 92, 4820–4824]. © 1990, American Chemical Society. All rights reserved.