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A Red-Shifted Chlorophyll

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Science  10 Sep 2010:
Vol. 329, Issue 5997, pp. 1318-1319
DOI: 10.1126/science.1191127

Abstract

Chlorophylls are essential for light-harvesting and energy transduction in photosynthesis. Four chemically distinct varieties have been known for the past 60 years. Here we report isolation of a fifth, which we designate chlorophyll f. Its in vitro absorption (706 nanometers) and fluorescence (722 nanometers) maxima are red-shifted compared to all other chlorophylls from oxygenic phototrophs. On the basis of the optical, mass, and nuclear magnetic resonance spectra, we propose that chlorophyll f is [2-formyl]-chlorophyll a (C55H70O6N4Mg). This finding suggests that oxygenic photosynthesis can be extended further into the infrared region and may open associated bioenergy applications.

Chlorophylls (Chls) are the essential pigments of photosynthesis, for which they both harvest light and transduce it into chemical energy. There are four chemically distinct chlorophylls known to date in oxygenic photosynthetic organisms, termed Chls a, b, c, and d in the order of their discovery (1, 2). All four pigments are present in light-harvesting complexes, though until recently only Chl a was thought to be indispensable for energy transduction in the photosystem reaction centers (3). This paradigm was challenged when Chl d, long considered an artifact since its discovery in 1943 (4), was shown to constitute up to 99% of all Chl in the cyanobacterium Acaryochloris marina (5). In this and related organisms, Chl d can replace Chl a in the photosystems of oxygenic photosynthesis, thereby extending to the red the spectrum of light that can be harvested for carbon fixation (6). Here we report yet another chlorophyll, which we designate Chl f (2), that absorbs even further to the red.

The morphological features of stromatolites provide a unique environment for specific but diverse cyanobacterial communities (7). We cultured a sample from Hamelin pool under near-infrared light (720 nm) (8). Analysis of a methanolic extract of stromatolites from Shark Bay, Western Australia, by high-performance liquid chromatography (HPLC) revealed a complex mixture of chlorophylls (Fig. 1A): In addition to a detectable amount of Chl a (peak 3) and bacteriochlorophyll a (peak B), there were trace amounts of Chl d and a new pigment, Chl f (peak 2 in Fig. 1A). The optical absorption spectrum of Chl f in neat methanol has a red-shifted QY transition [wavelength of maximum absorption (λmax) = 706 nm] compared to other chlorophylls and a blue-shifted Soret band (λmax = 406 nm) (Fig. 1, B and D). The room-temperature fluorescence emission of isolated Chl f is maximal at 722 nm (with excitation wavelength of 407 nm) (Fig. 1D), which is also considerably red-shifted compared to other Chls (9). Chlorophyll f appears to be made by a filamentous cyanobacterium (fig. S3) based on the 16S ribosomal RNA (rRNA) sequence of our purest enrichment III culture (see supporting text), which contained only Chl a and Chl f by HPLC analysis.

Fig. 1

(A) HPLC traces detected on the basis of QY absorbance (600 to 750 nm) of methanolic extracts from the stromatolite sample and from aerobic enrichment cultures I and II (see supporting material). Differences in retention times between the top and the two lower traces are due to different chromatographic solvent systems (CH3CN⋅CH3OH to CH3OH gradient above, CH3OH⋅H2O to CH3OH gradient below). (B) Spectral comparison of HPLC peaks of interest from enrichment culture I: peak 1 (retention time tr = 17.62 min) contains Chl b and Chl d, peak 2 (tr = 17.98 min) mainly Chl f characterized by its red-shifted Qy absorption band (706 nm), and peak 3 (tr = 20.05 min) Chl a. (C) Chemical structure of Chl f with comparison to other chlorophylls. (D) Absorbance and fluorescence emission spectra (λexc = 407 nm) of purified Chl f in methanol at room temperature. Contributions from a Chl a–like contaminant are indicated by stars.

We assigned the molecular formula of Chl f (C55H70O6N4Mg) by mass spectral analysis based on the molecular ion at 906 m/z (mass/charge ratio). Phytol (C20H38) was identified by the prominent fragment at 628 m/z (fig. S1B), and Mg as the central metal by the molecular ion of the pheophytin (Pheo) (884 m/z, C55H72O6N4). A formyl group was identified by 1H nuclear magnetic resonance (NMR) spectroscopy of Chl f [δ = 11.35 ppm (parts per million)] (fig. S2); the red-shifted QY optical transition (Fig. 1D) suggested substitution on rings A or C (Fig. 1C). The transformation of a methyl to a formyl moiety is a known modification of Chl a, producing Chl b (10). Because Chl d has a formyl group at C-3 (3), and differs in its properties, this leaves C-2 and C-12 as potential sites that comply with the mass spectrum. Substitution at C-2 is implicated by the methine pattern of the NMR spectrum (fig. S2): No methine resonates at δ > 10 ppm, which would be the expected chemical shift for the 10-H in the event of a neighboring 12-CHO group (11); moreover, the clustering of the C-5, C-10, and C-20 methine resonances at 9.86, 9.79, and 9.77 ppm is expected for C-2 substitution based on the known spectra of Chls a, b, and d and spectra simulated by density functional theory (DFT) (table S1). Substitution at C-2 is also supported by DFT calculations of the optical spectra, which predict a large red-shift of the QY band relative to Chl a for a 2-CHO group (Δλ = 37.6 nm), and, surprisingly, a blue-shift for a 12-CHO group (Δλ = −6.5 nm) (table S2). Therefore, we propose that Chl f is [2-formyl]-Chl a (Fig. 1C). Isolation of cultivable pure strains bearing Chl f will be essential to define the function of this intriguing chromophore.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1191127/DC1

Materials and Methods

SOM Text

Figs. S1 to S3

Tables S1 and S2

References

References and Notes

  1. The term “Chl e” has been used previously (1215), but these reports from more than 50 years ago only described the pigments vaguely and were not followed up with further characterization, so the nature of these pigments remains uncertain. These pigments had absorbance maxima that differed from that of the pigment described here. To avoid confusion, we propose to name the new pigment Chl f so as to maintain continuity with the nomenclature of the reported chlorophylls.
  2. Materials and methods are available as supporting material on Science Online.
  3. M.C. and B.A.N. hold Australian Research Council (ARC) fellowships and associated financial support. M.C. thanks K. Donohoe (University of Sydney) for assistance with polymerase chain reactions. M.C. and M.S. thank B. Crossett (University of Sydney) for mass spectral analysis. M.C. and H.S. thank A. Kwan and J. Mackay (University of Sydney) for NMR spectral analysis and A. W. D. Larkum (University of Sydney) for valuable discussions and continued support. R.D.W. acknowledges support from Macquarie University Strategic Infrastructure Scheme and Research and Development Scheme grants and the ARC and National Health and Medical Research Council (NHMRC) fluorescent applications for biotechnology in life sciences network for use of the Leica confocal imaging system. R.D.W. and M.C. thank D. Birch (Macquarie University) for help with the Olympus confocal imaging. Z.-L.C. thanks J. Reimers (University of Sydney) for supercomputer access. The 16S rRNA sequence has been deposited in GenBank under accession number 1370306.
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