Characterization of the primary electron donor of photosystem I, P700, by electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy

Abstract

Abstract The redox reactions of P700, the primary electron donor of photosystem I (PS I), have been analyzed by electrochemistry and Fourier transform infrared (FTIR) spectroscopy in two different preparations from the cyanobacterium Synechocystis PCC 6803 yielding monomeric and trimeric PS I complexes, respectively. Reversible and quantitative oxidation and rereduction of P700 has been achieved at surface-modified electrodes in an optically and infrared transparent thin-layer electrochemical (OTTLE) cell. The midpoint potential ( E m = +260 ± 10 mV vs. Ag/AgCl/3 M KCl) determined from these titrations, either by monitoring the electrochemically-induced oxidation/reduction of the primary electron donor P700 at 700 nm or by monitoring the amplitude of the flash-induced absorbance change at 707 nm as a function of the applied potential, agree between monomeric and trimeric PS I, demonstrating that protein-protein contact has very little impact on the redox properties of P700. Using electrochemical oxidation and rereduction, the vibrational IR difference spectra P700 ·+ − P700 could be generated. In the 1800−1200 cm −1 spectral region, highly structured IR difference spectra were obtained. The IR difference bands titrate in unison and are fully reversible upon application of a reducing potential. The midpoint potential measured by titration of the absorbance difference of two prominent bands, at 1714 and 1696 cm −1 ( E m = +260 ± 10 mV) corresponds to the values determined by titration of the electronic transition of P700. In contrast to previously obtained FTIR difference spectra for light-induced charge separation (E. Nabedryk, M. Leonhard, W. Mantele and J. Breton, Biochemistry, 29 (1990) 3242; G. MacDonald, K.A. Bixby and B.A. Barry, Proc. Natl. Acad. Sci. USA, 90 (1993) 11024) which include contributions from the primary electron donor and the acceptor side, the electrochemical generated FTIR difference spectra exclusively reflect the molecular changes at P700 and its protein site upon removal of an electron. Nevertheless, they largely agree with the difference spectra of the charge-separated state. In the 1800-1680 cm −1 range, the IR difference signals are attributed to the keto and ester C=O modes of P700. Between 1700 and 1680 cm −1 , shifts of bands and changes of band intensities for photosystem I particles suspended in 2 H 2 O reveal contributions of amide I modes. Further effects of 2 H 2 O/ 1 H 2 O exchange are observed between 1600 and 1500 cm −1 , and around 1400 cm −1 . Between 1750 cm −1 and approximately 3000 cm −1 , no FTIR difference signals were detected. In particular, no broad but strong absorbance changes were detected around 2600 cm −1 , where a low-energy electronic transition, characteristic for the dimeric nature of P and P + , was detected for P865 in bacterial reaction centers (J. Breton, E. Nabedryk and W. Parson, Biochemistry, 31 (1992) 7503). This indicates that the positive charge on P700 ·+ is rather localized on one of the chlorophylls.