18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

Martin Schliep,Ben Crossett,Robert D Willows,Min Chen

doi:10.1074/jbc.m110.146753

Abstract

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.

Highlights

Chl a, b, or c) do not absorb [3] and 2) it is the only Chl found so far that can substitute for Chl a in charge separation in the reaction centers of oxygenic photosynthetic organisms (4 – 6)
18O Labeling in Chlorophyll d Biosynthesis grown in the presence of H218O in the culture medium for 48 h background of unlabeled Chl d present at the start of the labelwas 900.44 m/z
The oxygen atom of the C3-acetyl group of BChl a is derived from water, and a similar reaction mechanism could be responsible for the incorporation of a; B, Chl d

Summary

Introduction

B, or c) do not absorb [3] and 2) it is the only Chl found so far that can substitute for Chl a in charge separation in the reaction centers of oxygenic photosynthetic organisms (4 – 6). One-ml cell culC31-formyl group oxygen atom of Chl d will be a crucial step ture aliquots were sampled for pigment extraction at 24, 48, 72, toward understanding the mechanisms of the biosynthetic 96, 150, and 200 h, respectively.

Results

Conclusion