1. Truth and the Scientific Method

As a present for the new year, back in 2008, a prophecy appeared as a peer reviewed pre-publication. In this paper it was predicted that arsenic would be found in the backbone of nucleic acids of living organisms, replacing the ubiquitous phosphorus. The prophecy, as is often the case with this type of beliefs, also suggested a place on Earth where this would happen: Lake Mono in California (Wolfe-Simon et al., 2008). On April 6th, 2008, this prophecy was communicated to the world by a popular science magazine (Reilly, 2008). Now, at the end of 2010, as a Christmas present (in Continental Europe, December 2nd), NASA issued a sensational press release announcing that, yes, the prophecy had come true, and not on an exotic planet, but on our old mother Earth and exactly at the place where this was predicted to happen (Wolfe-Simon et al., 2010).

In ancient times, the sayings of prophets were the norm, but this is not science. Some would accept the prophecies, some would be miscreants. One may dispute the demarcation between science and non-science, but, in any event, a core criterium to accept facts as belonging to science is to understand that we cannot get direct access to Truth (if it ever exists). We can only make models of Nature. In this process we must avoid trying to prove that the model is right, but, rather, try to find where models are an inadequate representation of Nature (Popper, 1972).

If this fairly standard way of proceeding (explicitly stated in the NIH guidelines, for example) had been followed, the Wolfe-Simon et al., (2010) should never have been published; and here the authors and the editors of the magazine Science (as well as those that let pre-publications of this work leak out since 2008) are squarely to blame for not only did they fail to perform the basic function expected of a scientific journal, but instead participated in a cheap publicity stunt, and along with the authors and NASA, ran after fame with a misleading "press release" followed by yet another misinformation campaign to impress the mass media and garner the support of the masses. However the authors may be granted extenuating circumstances as the pressure to publish in high impact journals has become almost unbearable for most scientists in our days.

To substantiate this contention I review here basic arguments that should have come to mind before publication: chemical consequences of placement of atoms in the periodic table of element; biochemical experiments that demonstrate fragility of modifications of the phosphodiester bond, and biochemical data documenting instability of arsenate derivatives of nucleotides; ability of living organisms to concentrate elements present in traces in the environment, and analysis of genomic data that suggest a biochemical process permitting cells to alleviate arsenic toxicity.

2. Life and the Periodic Table

Many ideas of what life could be have been discussed for millenia. The most imaginative authors envisioned forms of life distinctly alien and based on principles of life completely different from the life we know (see for example Hoyle, 1957). By contrast Wolfe-Simon et al., (2010) imagine a form of life that is based on principles that are identical to those governing our standard view of life, but with one big difference: arsenic would replace phosphorus in its construction (Wolfe-Simon et al., 2010). This places does raise basic questions, which, of course, have been asked before: why are the elements we find in living organisms in limited number, and why were they those specific elements essential to the mix?

The short answer is straightforward. Life develops around 300K, with water as its bathing medium. A core property of its components is that beside a limited number of small molecules (a few tens of atoms), it is made of macromolecules: giant polymers obtained by elimination of a water molecule between a small alphabet of basic building blocks, amino acids and nucleotides. This seemingly simple arrangement has a remarkable property in terms of information: while the backbone of these polymers is invariable, the side chains can be arranged in an infinite variety of combinations.

This informational view must be associated with physico-chemical constraints at the required temperature. Making molecules, and a fortiori polymers, implies forming stable covalent bonds. A covalent bond appears when electrons share their presence between two or more atoms’ nuclei. Now, the electrons associated to a nucleus are arranged along specific constraints ruled by quantum physics laws. And the consequence of these laws is that the various atoms of the universe can be arranged along rows and columns, according to the way they match their electrons with the charge of their nuclei. This constitutes the periodic table of elements (Figure 1).

As shown in the figure, other atoms are also involved in life. Most, in fact, are playing important roles in specific features of catalytic reactions needed to construct, modify and destroy covalent bonds, electrostatic interactions (metals) and more or less complicated electron exchanges (transition metals and complex anions such as molybdate or tungstate). Arsenic itself can be found in some rare arsenolipids.

Two exceptions remain, that have to be accounted for. Sulfur (the higher homolog of oxygen) and phosphorus (the higher homolog of nitrogen). The former, being in a deeper row than oxygen, is mainly involved in exchanges of electrons (its oxido-reduction potential varies from -2 to +6), in formation of energy-rich thioesters and via a remarkable affinity for iron, in making iron-sulfur clusters that have probably had a seminal role on the origin of life on Earth, where it is quite abundant (Wickramasinghe, 1973). The latter, phosphorus, is the candidate that Wolfe-Simon and colleagues proposed to see replaced by arsenic. This is not chemically possible, as we shall discuss now.

3. The nucleic acid phosphodiester backbone is fragile

Phosphorus, in living cells, is essentially used as phosphate, involved in three major processes: carrying and transporting energy, forming the backbone of nucleic acids, and acting as an energy-dependent tag for regulation. In fact the association of phosphorus with oxygen atoms, making the phosphate anion, has the property to make long chains (polyphosphates) when desiccated. These chains are, of course, prone to hydrolysis, but remarkably, in a highly metastable way. This means that while the stable forms are those which results from hydrolysis (the phosphate anions), to reach this stage, the compound needs to go through a very high activation energy barrier. This is the reason why the phosphate bond has been named energy-rich, since the discovery of the role of ATP by Fritz Lipmann (Lipmann, 1975). And this is why phosphate is the basic currency of energy in life. This metastability extends to the formation of phosphoesters, and in particular phosphodiester bonds. And this permitted the formation of nucleic acids, which use a negatively charged backbone to carry and protect from the environment fragile base pairs in a double helical structure. Life warns us already of the intrinsic fragility of these metastable bonds. Modifications of the (deoxy)ribose-phosphodiester backbone exist in nature, but they seldom affect the phosphate group. There is a modification, however, that is relevant in the present context. In Streptomyces lividans a DNA modification system replaces a side oxygen of phosphate by an atom of sulfur at some specific places of the DNA backbone (phosphorothioation (Zhou et al. 2005)).

This modification makes DNA unstable in vitro by oxidative, double-stranded, site-specific cleavage during normal and pulsed-field gel electrophoresis, making completion of genome sequencing a difficult task. A database of genomes possessing the same modification, which makes the completion of the corresponding genome sequencing difficult, has been published (Ou et al. 2009).

This modification, which was long unidentified, has some conceptual similarity to the phosphorous - arsenic swap, as sulfur belongs to the same family as oxygen in Mendeleiev’s table, but it is located at a much less crucial position in the backbone. It also tells us that beside hydrolysis, oxidation may be central in the instability of arsenic, if it were to replace phosphorus.

4. No arsenic involvement in formation of high energy bonds

But there is even more direct biochemical evidence that makes impossible for arsenic to play the role of a stable component of a nucleic acid backbone in an aqueous environment. Arsenic is a poison, and this has been known for millenia. Beside involvement in oxido-reduction reactions (which can be used both to evolve arsenite into its more innocuous form arsenate, and to recover energy), arsenic can indeed replace phosphorus in many phosphorolytic reactions and even form carbohydrate arsenates that are similar to their phosphate analogs (Lagunas & Sols, 1968). However this is limited to exchange reactions that cannot lead to stable and useful compounds.

It is also possible to begin to construct compounds along the line predicted to exist if phosphorus could replace arsenic, for example by constructing an arsenical analog of adenosine diphosphate (an essential pre-requisite if the if the claims published by Science were true). While difficult, a synthesis in which the phosphono-oxy group of ADP was replaced by the arsenomethyl group (arsenate itself was far too unstable at this position, requiring replacement of the bridging oxygen by a methylene group) was achieved by Dixon and colleagues. The product was unable to compete for ADP in any of its standard substrates (Webster et al. 1978). A further demonstration puts the final nail in the coffin: the arsonomethyl analogue of AMP can be used as a substrate for adenylate kinase. It permits transfer of a phospho group from ATP, but like all anhydrides of arsonic acids breaks down immediately, transforming the enzyme into an ATPase (Adams et al. 1984). The situation would be even worse if the hypothetical arsenical analog of ATP could have existed.

5. Living organisms know how to concentrate trace elements

Performing experiments with « pure » compounds is notoriously difficult. Many investigators have observed that Escherichia coli in media containing « pure » sodium salts will grow, albeit slowly and to a limited extent. This is not due to sodium replacing potassium but to a remarkable enrichment of potassium present in elusive traces. Another - unfortunately common - practice is to grow bacteria in synthetic media where common salts (potassium, magnesium and iron) are included, without any addition of essential metals such as zinc or manganese (Miller, 1992). Yet bacteria grow, and grow well. I do not suspect them to be able to transmute matter, or to universally substitute zinc or manganese by iron or magnesium. In the same way the well known « fluoride effect » results from extraction of aluminum from glassware, making AlF4, which, in fact, mimicks phosphate (Bigay et al. 1985).

In Wolfe-Simon and colleagues experiments the traces of phosphate present as contaminant at most steps in the building up of their growth medium, is certainly sufficient to permit growth of the cells, with phosphorus playing its normal role. The main question to answer, then, is not whether phosphate remains present with its standard role, but how the cells cope with an environment which is arsenic-replete.

6. Sulfur and arsenic: a likely mutual protective antagonism

The genome of several arsenic-loving bacteria has been sequenced, in particular that of Herminiimonas arsenicoxydans (Muller et al. 2007). This organisms uses arsenic to perform electron transfers that permit it to manage energy while alleviating some of its toxicity. But this cannot be enough, most probably, to detoxify this element. Analysis of the genome sequence, as well as that of other arsenophilic bacteria (including immediate kins of the Halomonas species used in Wolfe-Simon and colleagues’ study) suggests a remarkable conjecture to explain how it does so. While H. arsenicoxydans does not use selenium (as other bacteria such as E. coli do, to make selenocysteine, the 21st amino acid), it has the counterpart of the selD gene. I note that Halomonas elongata possesses the counterpart of this gene (HELO_4105). SelD in « standard » bacteria is the enzyme that makes selenophosphate, used to modify serine on a tRNA that translates UGA codons into selenocysteine.

Selenium is the counterpart of sulfur in Medeleiev’s table, it occupies a volume that is slightly larger than the latter. A similar situation is met with arsenic and phosphorous. Hence, a small distortion of the standard SelD catalytic site would easily accomodate sulfur instead of selenium and arsenate instead of phosphate. The role of SelD would then be to make monothioarsenate (Figure 2). This compound can react with a variety of compounds, biopolymers or minerals that it would decorate with arsenic in an innocuous form.

This hypothesis and others should be further explored before we propose that life as we know it can develop using arsenic in the place of phosphorus.

7. Conclusion

The idea that arsenic could have replaced phosphorus as a central component of nucleic acids should never have been published in a scientific journal. However, the authors should not bear the whole burden of the blame. The nature of science is to conduct experiments with proper controls and obtain results. To be communicated to other investigators these results need to be written up and submitted as an article to a scientific journal for peer review. Unfortunately, because of competition for limited funding a hierarchy has been progressively built up, with some journals considered as more important than others because of the impact they have on their readers. Lacking proper scientific training, many journalists tend to take the impact factors of journals as a proof of scientific quality. This is not so, unfortunately. And more often than not, as we see in the present situation, high profile journals failed in the basic responsibilities required of a scientific journal and then participated in a strange and misleading publicity campaign that fooled the public. In a context where there is a growing loss of trust in science and scientists, this will have most damaging consequences.

Acknowledgments: This work benefited from discussions with Philippe Marlière. It was supported by MICROME, a Collaborative Project funded by the European Commission within its FP7 Programme, contract number 222886-2.