The cyanobacterium Acaryochloris marina was cultured in the presence of either H(2)(18)O or (18)O(2), and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H(2)(18)O, newly synthesized Chl a and d, both incorporated up to four isotopic (18)O atoms. Time course H(2)(18)O labeling experiments showed incorporation of isotopic (18)O atoms originating from H(2)(18)O into Chl a, with over 90% of Chl a (18)O-labeled at 48 h. The incorporation of isotopic (18)O atoms into Chl d upon incubation in H(2)(18)O was slower compared with Chl a with approximately 50% (18)O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of (18)O(2) gas, one isotopic (18)O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H(2)(18)O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic (18)O atoms derived from molecular oxygen ((18)O(2)) was observed in the extracted Chl d, and the percentage of double isotopic (18)O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3(1)-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.
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