Abstract
This study reveals by in vivo deuterium labeling that in higher plants chlorophyll (Chl) b is converted to Chl a before degradation. For this purpose, de-greening of excised green primary leaves of barley (Hordeum vulgare) was induced by permanent darkness in the presence of heavy water (80 atom % (2)H). The resulting Chl a catabolite in the plant extract was subjected to chemical degradation by chromic acid. 3-(2-Hydroxyethyl)-4-methyl-maleimide, the key fragment that originates from the Chl catabolite, was isolated. High resolution (1)H-, (2)H-NMR and mass spectroscopy unequivocally demonstrates that a fraction of this maleimide fragment consists of a mono-deuterated methyl group. These results suggest that Chl b is converted into Chl a before degradation. Quantification proves that the initial ratio of Chl a:Chl b in the green plant is preserved to about 60-70% in the catabolite composition isolated from yellowing leaves. The incorporation of only one deuterium atom indicates the involvement of two distinguishable redox enzymes during the conversion.
Highlights
71-hydroxy Chl a in intact barley etioplasts, the reduction required NADPH or NADH
In this work we evince by spectroscopic methods that during the de-greening of barley leaves (H. vulgare) in heavy water a fraction of the Chl a catabolite is deuterium labeled; one deuterium atom is incorporated in the methyl group of the apparent chlorophyll a catabolite
The region of the methyl group is shown. a, this trace shows the long range J5 triplet coupling of the tri-protio-methyl group with the methylene group situated at C(31). b, Gaussian multiplication of the free induction decay before Fourier transformation and amplification of the signals in close proximity to the tri-protio-methyl group unveils the fraction of molecules having a geminal 1H/2H coupling. c, result of a double resonance experiment in which the methylene group C(31) at ␦ ϭ 2.67 ppm was irradiated
Summary
Chlorophyll; Chlid, chlorophyllide; Pheo, pheophorbide; AcOEt, ethyl acetate; AcMe, acetone; AcOH, acetic acid; CH2Cl2, dichloromethane; EtOH, ethanol; MeOH, methanol; dn, hn, number of attached deuterium or hydrogen atoms in a molecule; ppm, parts per million; MS, mass spectroscopy; amu, atomic mass units. In vivo labeling experiments with heavy water to elucidate biogenesis mechanisms are barely mentioned in the literature. The method has been applied to study peripheral changes of the porphyrin system during the biosynthesis of bacteriochlorophyll a in the photosynthetic bacterium Rhodospirillum rubrum [9, 10] and to follow the insertion of a deuteron during the light induced cyclization of dark synthesized acyclic carotenoide precursors in the green alga Senedesmus obliquus [11]. We have demonstrated by in vivo deuterium labeling experiments with C. protothecoides that in the last step of the macrocyclic ring cleavage a hydrogen atom is highly stereoselectively inserted in the catabolite [12]. In this work we evince by spectroscopic methods that during the de-greening of barley leaves (H. vulgare) in heavy water a fraction of the Chl a catabolite is deuterium labeled; one deuterium atom is incorporated in the methyl group of the apparent chlorophyll a catabolite. The results suggest that Chl b is converted to Chl a by two different cofactors
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