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

摘要

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 2H labeled, and uniformly 15N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D2O. Previously, we have found that extensive washing and incubation of PS I samples in D2O does not alter the (P700+-P700) FTIR difference spectrum, even with ∼50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform 2H labeling of PS I, however, the (P700+-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D2O and H2O, a (1H-2H) 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 2H 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 2H labeled PS I particles indicates that a negative difference band at 1698 cm−1 is associated with at least two species. The observed 15N and 2H induced band shifts strongly support the idea that the two species are the 131 keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at ∼1639 cm−1 is somewhat modified in intensity, but unaltered in frequency, upon 2H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.