Primary Donor Photo-Oxidation in Photosystem I:  A Re-Evaluation of (P700⁺ − P700) Fourier Transform Infrared Difference Spectra

Abstract

Light-induced Fourier transform infrared (FTIR) difference spectroscopy has been used to study the photo-oxidation of the primary electron donor (P700) in PS I particles from Chlamydomonas reinhardtii and Synechocystis sp. PCC 6803. To aid in the interpretation of the spectra, PS I particles from a site-directed mutant of C. reinhardtii, in which the axial histidine ligand (HisA676) was changed to serine, were also studied. A high-frequency (3300-2600 cm(-1)) electronic transition is observed for all PS I particles, demonstrating that P700 is dimeric. The electronic band is, however, species-dependent, indicating some differences in the electronic structure of P700 and/or P700(+) in C. reinhardtii and Synechocystis sp. 6803. For PS I particles from C. reinhardtii, substitution of HisA676 with serine has little effect on the ester carbonyl modes of the chlorophylls of P700. However, the keto carbonyl modes are considerably altered. Comparison of (P700(+) - P700) FTIR difference spectra obtained using PS I particles from the wild type (WT) and the HS(A676) mutant of C. reinhardtii indicates that the mutation primarily exerts its influence on the P700 ground state. The 13(1) keto carbonyls of the chlorophylls of P700 of the wild type absorb at similar frequencies, which has previously made these transitions difficult to resolve. However, for the HS(A676) mutant, the 13(1) keto carbonyl of chlorophyll a or chlorophyll a' of P700 on PsaB or PsaA absorbs at 1703.4 or 1694.2 cm(-1), respectively, allowing their unambiguous resolution. Upon P700(+) formation, in both PS I particles from C. reinhardtii, the higher-frequency carbonyl band upshifts by approximately 14 cm(-1) while the lower frequency carbonyl downshifts by approximately 10 cm(-1). The similarity in the spectra for WT PS I particles from C. reinhardtii and Synechocystis sp. 6803 indicates that a similar interpretation is probably valid for PS I particles from both species. The mutant results allow for an interpretation of the behavior of the 13(1) keto carbonyls of P700 that is different from previous work [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585-11592], in which it was suggested that 13(1) keto carbonyls of P700 absorb at 1697 and 1639 cm(-1), and upshift by 21 cm(-1) upon cation formation. The interpretation of the spectra reported here is more in line with recent results from ENDOR spectroscopy and high-resolution crystallography.

Mutation Induced Modulation of Hydrogen Bonding to P700 Studied Using FTIR Difference Spectroscopy

“…The mutation induced upshift of the positive 1686.4 cm -1 band in the WT spectrum to 1688.7 cm -1 in the mutant can also explain, at least in part, the negative band at 1690.4 cm -1 in the FTIR DDS in Figure 5C . This again is compatible with our previous interpretation of FTIR DS (14).…”

“…Previously, we suggested that the ∼1637(-)/1654 cm -1 difference band in WT FTIR DS ( Figure 5A) is due to both HisA676 and HisB656. More specifically, the difference band is due to the C 4 dC 5 modes of the histidine imidazoles that are protonated at one nitrogen and involved in metal binding at the other (14). The shoulder observed at 1632.0 cm -1 in the WT FTIR DS in Figure 5B supports the idea that at least two negative components contribute to the ∼1637(-) cm -1 difference band.…”

“…FTIR spectra were recorded using a Bruker IFS/66 FTIR spectrometer. Spectra were collected before, during, and after light excitation from a helium-neon laser, as described (14). All spectral analyses (subtractions, second derivative calculations, Fourier deconvolution, and curve fitting) were performed using the software package OPUS, supplied by Bruker Instruments.…”

“…The origin of the 1688(+) cm -1 band is not considered in this model. In an alternative interpretation of WT (P700 + -P700) FTIR DS, we proposed that the 1700(-) cm -1 band is due to the 13 1 keto CdOs of both P B and P A , at ∼1703 and ∼1697 cm -1 , respectively (14). The ∼1703 cm -1 band of P B upshifts to ∼1716 cm -1 upon cation formation, while the ∼1697 cm -1 band of P A downshifts to ∼1687 cm -1 (14).…”

“…In an alternative interpretation of WT (P700 + -P700) FTIR DS, we proposed that the 1700(-) cm -1 band is due to the 13 1 keto CdOs of both P B and P A , at ∼1703 and ∼1697 cm -1 , respectively (14). The ∼1703 cm -1 band of P B upshifts to ∼1716 cm -1 upon cation formation, while the ∼1697 cm -1 band of P A downshifts to ∼1687 cm -1 (14). We also suggested that the 1636(-)/1654(+) cm -1 difference band is due to modes associated with the axial histidine ligands (HisA676 and B656 in C. reinhardtii).…”