Wang Ruili, Sivakumar Velautham, Johnson T Wade, Hastings Gary
Department of Physics and Astronomy, Georgia State University, Atlanta, Georgia 30303, USA.
Biophys J. 2004 Feb;86(2):1061-73. doi: 10.1016/s0006-3495(04)74181-8.
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.
利用来自集胞藻6803的光系统I（PS I）颗粒，已获得室温下光诱导的（P700(+) - P700）傅里叶变换红外（FTIR）差谱，这些颗粒未标记、均匀（2）H标记和均匀（15）N标记。还获得了在D(2)O中广泛洗涤和孵育后的PS I颗粒的光谱。此前，我们发现即使有大约50%的质子交换，在D(2)O中对PS I样品进行广泛洗涤和孵育也不会改变（P700(+) - P700）FTIR差谱。这表明溶剂水无法进入P700结合位点。然而，当对PS I进行均匀（2）H标记时，（P700(+) - P700）FTIR差谱会发生显著变化。从使用在D(2)O和H(2)O中生长的PS I颗粒获得的光谱中，构建了一个（（1）H - （2）H）同位素编辑双差谱，结果表明，与P700和P700(+)叶绿素的酯/酮羰基模式相关的所有差谱带在（2）H标记后分别下移4 - 5/1 - 3 cm(-1)。还表明P700叶绿素的酯和酮羰基模式在频率上无需非均匀分布。最后，我们在差谱中未发现半胱氨酸模式存在的证据。使用（2）H标记的PS I颗粒获得的光谱表明，1698 cm(-1)处的负差谱带与至少两种物质相关。观察到的（15）N和（2）H诱导的谱带位移有力地支持了这两种物质是P700两种叶绿素的13(1)酮羰基模式的观点。我们还表明，大约1639 cm(-1)处的负差谱带在（2）H标记后强度有所改变，但频率不变。这表明该谱带与P700一种叶绿素的强氢键酮羰基模式无关。
