An experimental study of magnesium-isotope fractionation in chlorophyll-a photosynthesis

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Abstract

Measurements are presented of the magnesium isotopic composition of chlorophyll-a, extracted from cyanobacteria, relative to the isotopic composition of the culture medium in which the cyanobacteria were grown. Yields of 50–93% chlorophyll-a were achieved from the pigment extracts of Synechococcus elongatus, a unicellular cyanobacteria. This material was then digested using concentrated nitric acid to extract magnesium. Separation was accomplished using columns of cation-exchange resin, which achieved a 103 ± 10% yield of magnesium from chlorophyll-a. This procedure ensured accurate measurement of the magnesium-isotopic ratios without isobaric interferences using a multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). We find a slight depletion in the heavier isotopes of magnesium in chlorophyll-a relative to culture medium, early growth phase: Δ26Mg = −0.71(±0.35)‰ and Δ25Mg = −0.37(±0.18)‰; late growth phase: Δ26Mg = −0.53(±0.20)‰ and Δ25Mg = −0.26(±0.11)‰, due to an apparent mass-dependent fractionation. We suggest that the small fractionation results from chelation during intracellular processes. A likely candidate for this chelation step involves the magnesium-chelatase enzyme, which mediates the insertion of magnesium to the tetrapyrrole ring during chlorophyll-a biosynthesis. Proof of this hypothesis can be tested with biological controls whereby steps in the enzymatic pathways of chlorophyll synthesis are selectively suppressed.

Introduction

Rates of energy conversion by photosynthesis across the planet are enormous and account for ∼105 gigatonnes of carbon per year (Field et al., 1998). This primary production is important to all geochemical cycles. Chlorophyll-a is frequently used as a marker of biomass (e.g., Falkowski et al., 1998, Roman et al., 2005) and the global distribution can be monitored by satellite. Magnesium is the metal center in the chlorophyll molecule and thus is central to all photosynthesis, and hence to the history and geochemistry of life on the planet.

In this paper, we test the hypothesis that magnesium isotopes fractionate during biosynthesis of chlorophyll-a, one of the most common types. The ubiquity of the naturally occurring isotopes of magnesium, 24Mg (78.992%), 25Mg (10.003%) and 26Mg (11.005%) leads us to the question: ‘Is there a fractionation of these isotopes during the synthesis of chlorophyll and could this be used as a potential biomarker?’

The potential geochemical significance of this fractionation is considerable, since the fractionation may be affected by temperature or other environmental conditions and thus the isotopic composition of chlorophyll may provide a record of geochemical conditions. There is some evidence in the literature of isotopic fractionation in both chlorophyll-a and -b (Galy et al., 2001). Young and Galy (2004) report δxMg values (part per thousand variations, x = 25, 26, see Eq. 1) relative to the DSM3 standard of δ26Mg = −1.451‰, δ25Mg = −0.741‰ for spinach chlorophyll-a, and δ26Mg = −2.349‰, δ25Mg = −1.204‰ for spinach chlorophyll-b. However, the magnesium-isotopic composition of the culture medium from which the spinach was grown was not known, as they used chlorophylls from a commercial source for their study. Similarly, Ra et al. (2003) report a large difference in the δ26Mg of chlorophyll-a and -b from several commercial sources, including cultured planktonic species and marine phytoplankton samples, but did not detail the data in their abstract.

It is reasonable to expect a magnesium-isotope fractionation given our current understanding of the biosynthetic pathways of chlorophyll. Fractionation could take place during the insertion of magnesium into protoporphyrin IX (Fig. 1), which involves the magnesium-chelatase enzyme (e.g., Willows, 2003). This enzyme binds the Mg(II) tightly and introduces it into the porphyrin in a late state. Similar chelating resins and magnesium-binding molecules are known to induce an isotopic fractionation in purely inorganic systems (e.g., 26Mg enrichment on functionalized Merrifield peptide resins, Kim and Kang, 2001, Kim et al., 2002, Kim et al., 2003).

Recent studies by Buchachenko, 2001, Buchachenko et al., 2005a, Buchachenko et al., 2005b, suggest a wholly different kinetic pathway that may involve coupling of the electron- and nuclear-spins in a free-radical reaction. The essence of this unconventional pathway is that decay of a transient free radical proceeds much faster (e.g., 2–3 times for ATP synthesis, Kuznetsov et al., 2005) in the presence of magnetic 25Mg than diamagnetic 24Mg (Buchachenko et al., 2005a). The difference is entirely kinetic and relates to the relative rates of relaxation of excited electrons in the vicinity of the nuclear spin. If a kinetic advantage is to be gained by concentrating the magnetic isotope, one wonders if the enzymatic pathways concentrate 25Mg. If so, a mass-independent magnesium-isotope fractionation is expected.

Section snippets

Growth of cyanobacterial cultures

A culture of Synechococcus elongatus (Anacystis nidulans, PCC 7942), a unicellular cyanobacteria, was provided by Prof. John Meeks at the University of California, Davis. This cyanobacteria was chosen for this study because it produces only chlorophyll-a, thus eliminating the need to separate chlorophyll-a and -b from the pigment extract. Samples of cyanobacteria were grown in a freshly prepared BG11 culture medium (Table 1) by dissolving ∼1.65 g in 1 L of water (18 MΩ resistance) and adding 1 ml

Results

The ratios of 26Mg/24Mg and 25Mg/24Mg measured using the MC-ICP-MS were standardized against the international standard DSM3 by converting these values to a δxMg:δxMg=(xMg/24Mg)sample(xMg/24Mg)DSM3-1×1000where x = 26 or 25.

The results, and a comparison with other literature studies, are summarized in Table 3, which reports the average δxMgDSM3 value from a number of replicate measurements of the samples and standards. For DSM3, the δxMgDSM3 value is 0 by definition. Table 3 also reports the

Discussion

The simplest interpretation of these experiments is that the fractionation of magnesium isotopes occurs in growth of the cyanobacteria and synthesis of chlorophyll-a. Our results show a depletion in the heavier isotopes of magnesium, early growth phase: Δ26Mg = −0.71(±0.35)‰ and Δ25Mg = −0.37(±0.18)‰; late growth phase: Δ26Mg = −0.53(±0.20)‰ and Δ25Mg = −0.26(±0.11)‰, relative to the culture medium (Fig. 5).

The step that causes fractionation in chlorophyll synthesis is unknown, but we suggest that it

Conclusions

The magnesium-isotopic values that we report here for chlorophyll-a, in an early growth phase, are similar to those reported by Young and Galy (2004), all relative to the DSM3 standard. The isotopic fractionation between the culture media and magnesium extracted from chlorophyll-a is shown for the first time (early growth phase: Δ26Mg = −0.71(±0.35)‰ and Δ25Mg = −0.37(±0.18)‰; late growth phase: Δ26Mg = −0.53(±0.20)‰ and Δ25Mg = −0.26(±0.11)‰). We suggest that the depletion of heavier magnesium

Acknowledgments

We thank Professors David Britt, John Meeks and Albert Galy for providing advice and material for these experiments. We thank Benjamin Jacobsen for his assistance in the MC-ICP-MS laboratory. The isotopic measurements were made at the UC Davis Interdisciplinary Center for Plasma Mass Spectrometry (UCD-ICP-MS) funded by NSF and UC Davis. This work is contribution No. 0012 from the UCD-ICP-MS. This research was supported by the US DOE Office of Basic Energy Sciences via Grant DE-FG03-02ER15325 to

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