FTIR Difference Spectroscopy in Combination with Isotope Labeling for Identification of the Carbonyl Modes of P700 and P700+ in Photosystem I

Abstract

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.

Rapid Determination of Chlorophyll and Pheophytin in Green Tea Using Fourier Transform Infrared Spectroscopy

“…The main large peaks of 1650, 1525, 1458, 1239, 1150, and 1039 cm −1 could be detected in the fingerprint region of the FT–IR spectra. In detail, the peak around 1650 cm −1 could be the vibration of the amide I (N–H bending), the peak around 1525 cm −1 is related to the band of the amide II, and the peak of 1239 cm −1 is assigned to the amide III band [ 23 , 24 ]. The peak at 1150 cm −1 is assigned to the anti-symmetric stretching of the C–O–C bridge [ 25 ].…”

Different Structural Changes Occur in Blue- and Green-Proteorhodopsins during the Primary Photoreaction

“…The amide II normal mode of the peptide backbone could contribute to this region as shown recently in the sub-picosecond IR difference spectrum of GPR at room temperature (32). To distinguish between these possibilities, we labeled the retinal with a deuterium on carbon 15 (C15D) which shifts the ethylenic frequency ~5 cm −1 (33) and separately grew the protein on total- 15 N labeled media which shifts the amide II mode ~15 cm −1 (32, 34, 35). As shown in Figure 2, in BPR regenerated with C15D retinal, the negative band at 1557 cm −1 downshifts to 1549 cm −1 while the positive band at 1529 cm −1 downshifts to 1525 cm −1 .…”

Fourier transform infrared spectroscopy of special pair bacteriochlorophylls in homodimeric reaction centers of heliobacteria and green sulfur bacteria

“…PCC 6803, this signal was not found in P700 ? /P700 spectra by other groups (Breton et al 1999;Wang et al 2004;Noguchi et al, unpublished data), and hence could be due to some artifact. Oh-oka (2007) suggested that PshA-C601, which is buried in the interior of membranes and located near the His ligand to P798, is a most probable candidate for this signal.…”