Absorption Spectra of Pigments and Chromatophores ...

Absorption Spectra of Pigments and Chromatophores under Different Oxygen Concentrations

To study the effects of oxygen concentration on the synthesis of pigments（bacteriochlorophyll and carotenoids）and on light-harvesting proteins（LH-Ⅰ and LH-Ⅱ），R.capsulatus SB1003 was grown photosynthetically for 22 h to reach a density of 5×108 cells per ml.These logarithmically growing photosynthetic cells were designated PS.Samples of PS cells were immediately poured into two Erlenmeyer flasks covered with two layers of gauze；one was shifted to low oxygen（medium/flask ratio，8∶10，vol/vol）designated as LO2，and the other was shifted to high oxygen（medium/flask ratio，0.8∶10，vol/vol）designated as O2.Both were continuously cultured with vigorous shaking for 24 h.Steady-state high aerobic cells were obtained by inoculating oxygen-adapted cells into fresh RCV medium（medium/flask ratio，0.8∶10，vol/voL and culturing them aerobically in a shaker for 18 h to reach a similar density（5×108 cells per ml）for comparison.After 24 h，cells shifted from photosynthetic to LO2 conditions has grown only slightly because of a long period of adaptation，whereas cells shifted to O2 doubled in cell density after adaption to the O2 condition.The aeration of cells is directly correlated with their pigmentation：photosynthetic growth yields cells which are yellowish brown，LO2 conditions result in a purplish-redculture，O2 conditions yield yellowish-pink cells，and steadystate aerobic growtn gives rise to yellowish cells.The difference in colors of cell cultures results from the difference in the relative content and composition of carotenoids and bacteriochlorophyll.This is shown by the absorption spectra of the extracted pigments from the four types of cell cultures（see Figure 2）.The pigments from PS cells show absorption peaks typical of bacteriochlorophyll at 360，600，and 770 nm and peaks typical of carotenoids at 427，453，and 484 nm.Bacteriochlorophyll content gradually diminished after the cells were shifted to LO2 or O2 and was present only in trace amounts in steady-state aerobic cells.The decrease in amount of bacteriochlorophyll was correlated to oxygen concentration.The carotenoid content was also reduced about 50%when the cells were shifted to the LO2 or O2 conditions for 24 h.Carotenoids were present in steady-state aerobic cells（O2）at about 25%of the level in PS cells based on the area under the absorption bands between 400 and 500 nm.However，the amount of carotenoid relative to bacteriochlorophyll increased under high oxygen.

A more detailed analysis of the change in carotenoid content after the oxygen shift reveals that the amounts of carotenoid increased during the first hour by about 10%and then decreased over 24 h（data not shown）.It is interesting to note that the composition of carotenoids altered during the shift，as indicated by changes in the shape of the absorption peaks（see Figure 2）.The PS cells had a major absorption peak at 453 nm，a second peak at 484 nm，and a minor peak at 427 nm，all due to carotenoids.After the cells were exposed to oxygen（LO2 or O2），the peak at 427 nm disappeared，and the peak at 484 nm predominated，although all peaks became more rounded.Those three peaks were not separated in steadystate aerobic cells.The changes in the absorption peaks of carotenoids at 427，453，and 484 nm were detectable even 10 min after the cells were exposed to oxygen（data not shown）and gradually became more significant with time（from 0 to 24 h）（see Figure 3）.Such changes in absorption by carotenoids indicate that the composition of carotenoids underwent a significant alteration when the PS cells were exposed to oxygen.This conclusion was verified directly by thin-layer chromatography（data not shown）.Based on published thin-layer chromatography and mass spectroscopy analyses［28］，we have found that the major carotenoid of the PS cells was spheroidene.After the PS cells were shifted to LO2 or high O2 conditions，spheroidene was converted to spheroidenone.This conversion was correlated with oxygen concentration.The steady-state aerobic cells had much less total carotenoids；spheroidenone was the major carotenoid under these conditions.In agreement with the pigment absorption spectra，we have also observed by thin-layer chromatography that the amount of carotenoid relative to bacteriochlorophyll increased after the cells were shifted to high oxygen.

These results were also demonstrated by measurement of absorption spectra of chromatophores from cell lysates（see Figure 4）.The chromatophores had absorption peaks at 375，510，800，and 855 nm due primarily to bacteriochlorophyll complexed to lightharvesting proteins and at 450，480，and 510 nm due to carotenoids.After the PS cells were shifted to aerobic conditions，the absorption by carotenoids in chromatophores was reduced and the shape ofthe peaks was altered as for the extracted pigments.The amount of carotenoid relative to LH-Ⅰ and LH-Ⅱ，however，was found to increase in response to high oxygen.LH-Ⅰ LH-Ⅱ complexes，as measured by bacteriochlorophyll absorption peaks at 870 and 800 to 850 nm，respectively，also diminished after shifting to L02 or 02，and they were absent in steadystate aerobic cells except for a minor peak attributable to LH-Ⅰ at 870 nm.The peak at 855 nm is composed of bacteriochlorophyll absorption from both the LH-I and the LH-Ⅱcomplexes.The decrease in the LH-Ⅰ and LH-Ⅱ complexes in response to oxygen may result from the decreased biosynthesis and dilution of the existing LH pool by cell division.