December 2016 Issue Table of Contents
Life as we know it is completely dependent on metal ions. Gradients of metal ions drive metabolism, metal centers often form the active sites of enzymes, and metal-ion coordination is largely responsible for protein and RNA folding. This dependence on metal ions likely reflects the environment from which cellular life emerged. However, long chain biological polymers were not present on prebiotic Earth. Therefore, the chemical reactions leading to Earth’s first cells must have made use of alternative catalysts that were later superseded by RNA and protein. Here, we discuss the similarities between free metal ions, minerals, and biological enzyme catalysts, and how cellular life could have exploited prebiotic metallocomplexes.
Keywords: prebiotic chemistry, iron–sulfur cluster, protocell, metallopeptide
Darwinian evolution began on Earth approximately 3.5 billion years ago through a process that transformed geochemically driven organic synthesis and metabolic-like reactions into biochemistry. What exactly transpired may never be known because fossil records of protocellular life have yet to be found. Nevertheless, we do know that the principles of physics and chemistry are unchanged between geological and biological settings. In other words, Stanley Miller’s pioneering experimental work in the 1950s helped popularize the idea that the prebiotic components of life on early Earth could be synthesized in laboratory simulations. Since then, several laboratory experiments have shown that, in addition to amino acids, lipids and nucleotides can emerge from model prebiotic reactions. However, the mechanism by which individual monomeric units of amino acids and nucleotides could assemble into polymers similar to modern day proteins and nucleic acids has been investigated to a lesser extent than the formation of the monomers themselves. Nucleotides can polymerize in the absence of catalysts, and minerals can facilitate the polymerization of activated nucleotides (Orgel and Lohrmann 1974) and of amino acids (Kawamura et al. 2011). Furthermore, minerals can catalyze the formation of vesicles (Sahai et al. 2016 this issue), suggesting a scenario where minerals bring together nucleic acids and lipids to form cell-like compartments, although alternative scenarios are also possible.
Of all the biological polymers, RNA is the most versatile (Maurel and Leclerc 2016 this issue; Sahai et al. 2016 this issue). The ability of RNA to function as a genome and to catalyze chemical reactions makes RNA an ideal candidate for driving early evolution. This has led to investigations of RNA-containing model protocells as a way of gaining insight into the emergence of primitive cellular life (Kee and Monnard 2016 this issue). The model protocells can seemingly copy RNA, grow, divide, acquire nutrients, and compete for survival.
Investigations of RNA have revealed much about the chemical–physical underpinnings of cellular life, but much remains largely unexplored. For example, it is unclear how model protocells similar to those that have been built in the laboratory could incorporate a supporting metabolism, i.e. chemical reactions that maintain living systems. Also, RNA-catalyzed reactions are not known in the central metabolic reactions of contemporary biology (Keller et al. 2014). Instead, these reactions are mediated by protein enzymes: the digestion of food is mediated by proteins, not RNA. Although synthetic catalytic RNA molecules built in the laboratory are capable of accelerating a wide variety of reactions, catalytic RNAs are typically incapable of mediating the types of metabolic reactions found in biology. Explaining why this is so is important because, without a supporting metabolism, early cellular life would not have persisted for long enough to have evolved. It seems that some form of metabolism would have been needed early on, in part, to cope with the consumption of resources. In contrast, life is found today nearly everywhere on and near the Earth’s surface because organisms carry with them adaptable metabolic machinery to gather dispersed and varied foodstuff.
If RNA molecules did not catalyze all of the necessary chemical reactions, and protein enzymes were not present on prebiotic Earth, then what could have served as a prebiotic catalyst? Perhaps the required chemistry was mediated by minerals. This suggestion is attractive because some protein enzymes of contemporary life coordinate metallocomplexes that are similar to the constituents of minerals. For example, there is strong structural similarity between greigite (Fe3S4) and the Fe3S4 and Fe4S4 clusters found coordinated to several of the protein enzymes necessary to support life (Russell and Martin 2004).
The influence of metal ions on prebiotic chemistry may not have been confined to mineral surfaces. All organisms are heavily dependent on metal ions acquired from the environment. In fact, estimates put the number of metalloproteins somewhere between one-third and one-half of all proteins. The Fe3S4- and Fe4S4-containing proteins mentioned above are only a small fraction of the large number of metalloproteins needed to support contemporary life. Protein and RNA folding are largely dependent on metal-ion coordination and much of central metabolism is mediated by metal ions bound to proteins. However, the way metal ions could have been used to catalyze metabolic-like reactions prior to the existence of highly evolved proteins remains unclear.
Contemporary metalloproteins may be a consequence of prebiotic metal catalysts
Some of the chemical reactions prevalent on prebiotic Earth could have laid the foundation for modern metabolism (Morowitz 2004). Modern day protein enzymes facilitate and control chemical reactions that could initially have occurred abiotically. Not only were the same or similar reactions likely present on prebiotic Earth, but the reactions may have been favored over other competing reactions by the presence of prebiotic catalysts (Copley et al. 2007).
The identity of the prebiotic catalysts that shaped the emergence of contemporary metabolism may be gleaned from the protein enzymes that coordinate metal ions. This is because metal ions themselves, in the absence of protein or RNA, can function as catalysts—polymer scaffolds are not necessary. For example, free iron ions can catalytically decompose hydrogen peroxide, and several divalent cations alone can catalyze transphosphorylation reactions (Lowenstein 1958). Metal ions can form more complex, inorganic structures in the presence of compatible ligands. Iron–sulfur clusters analogous to those found in the redox active protein ferredoxin readily form in solutions of iron ions, inorganic sulfide, and additional sulfur ligands (Fig. 1). The resulting non–protein coordinated iron–sulfur cluster is catalytically active (McMillan et al. 1979).
Calvin (1959) noted that the ability of free Fe3+ to catalyze the degradation of hydrogen peroxide to water and oxygen was increased 1,000-fold when the iron was coordinated to porphyrin. An additional 107-fold improvement resulted from coordination of the iron porphyrin (heme) to the large protein enzyme catalase (Fig. 2). Although several intermediate steps would have had to transpire for a reaction catalyzed by free ferric ions to be transformed into a protein enzyme–mediated process, this example illustrates how the intrinsic activity of a metal center can be augmented by genetically encoded material. But it may not just be a case of evolution improving upon the innate activity of metal ions. The prevalence of metalloenzymes may reflect a difficulty in evolving metal-independent sequences, at least with the nucleotides and amino acids exploited by life as we know it. For example, in vitro evolution methods used to identify catalytic sequences invariably select for RNAs and proteins with metal-dependent activity, suggesting that sequence space is more sparsely populated with metal-independent folds. In other words, although enzyme-active sites devoid of metal centers can be envisaged (Casareno et al. 1995), evolution is more likely to hit upon a metal-dependent solution.
A protein’s metal content may reflect the environment from which the protein emerged
Attempts at determining the relevance of model prebiotic metallocomplexes must take into consideration thermodynamic affinity and availability (concentration), in addition to reactivity. The metal ions most exploited by biology are those that are readily found in the environment at a concentration of at least 1 nM (Egami 1974), perhaps reflecting a difficulty in forming binding sites with greater affinity in the cases of proteins and RNA ligands. The relationship between physiological relevance and availability is apparent when comparing the affinities of different metal ions for a specific metalloenzyme. That is, affinities typically correspond to the available concentration of the metal ion. For example, even if protein enzymes that mediate phosphorylation reactions frequently display increased catalytic activity in vitro with bound Mn2+, the affinity for this metal ion is not sufficient to result in coordination under physiological conditions. Instead, the affinity for Mg2+, the physiologically relevant metal, matches the availability of the metal ion (Cowan 1998). Once a protein has evolved to coordinate a metal ion, there is usually no selective pressure to increase the affinity.
Experimental evidence suggests that modern metabolism was shaped by free metal-ion catalysts. Keller et al. (2014) showed that the majority of the reactions of two fundamental metabolic pathways for the breakdown of sugar molecules (glycolysis and the pentose phosphate pathway) could be catalyzed by Fe2+ at elevated temperatures. Of the 10 steps of modern glycolysis, five Mg2+- and one Zn2+-dependent enzyme(s) are exploited (Fig. 3). Dissolved iron ions may have played an even larger role than the analysis of contemporary enzymes suggests. Athavale et al. (2012) demonstrated that Mg2+-dependent catalytic RNA activity can be recovered in the absence of Mg2+ by the addition of Fe2+. This latter finding is telling because under the more reducing conditions of prebiotic Earth, the availability of dissolved iron would have been greater than today due to the increased solubility of Fe2+ over Fe3+ and, thus, may have been preferentially used for catalysis. If true, then 60% of the steps of an early glycolytic-like pathway could have depended on iron ions. The impact of dissolved iron could have been even greater at an early evolutionary stage because the progenitors of contemporary nicotinamide adenosine dinucleotide [NAD(H)]-proteins may have coordinated iron or iron–sulfur clusters in place of the nicotinamide cofactor (Daniel and Danson 1995).
Contemporary organisms make use of two additional pathways to boost the energy yields from the breakdown of sugars, lipids, and proteins. These two pathways are called the citric acid cycle and the electron transport chain. The proteins of the citric acid cycle and electron transport chain are different from the proteins used in glycolysis in at least one aspect. The enzymatic steps of the citric acid cycle and electron transport chain are predominantly mediated by more complicated metallocomplexes, such as iron–sulfur clusters and heme, than those found in glycolysis. It is unclear if this dissimilarity in metallocomplexes for the different metabolic cycles reflects differences in the environment from which the proteins originated. In the absence of sulfide, an iron–sulfur cluster cannot form. Therefore, mononuclear Fe2+ coordination could have emerged from such conditions and may have been later substituted with Mg2+ when iron ions were less readily available, e.g. as described above for glycolysis. If, however, the environment did contain sulfide, then the formation of a more complex iron–sulfur cluster would have been more favorable. Because Mg2+ cannot form an analogous structure, the presence of the iron–sulfur cluster would have persisted, as seen as seen for the proteins of the extant citric acid cycle and the electron transport chain.
The potential effects of free metal ions on metabolic-like reactions is similar to previous theories based on mineral catalysts because the metal components of the minerals are critically important for activity. Although the only nominal metal in montmorillonite is aluminum, several metal-substituted species occur naturally as a consequence of local environments. In fact, only the alkali and alkaline earth metal substitutions of montmorillonite are able to polymerize nucleotides (Ferris 2005). Similarly, sodium substitution in montmorillonite aids in alanine adenylate polymerization (Paecht-Horowitz 1978), and the sodium forms of kaolinite and bentonite are able to polymerize glycine during wetting–drying cycles (Lahav et al. 1978). That is not to say that minerals are the same as free metal ions in terms of their potential as prebiotic catalysts. In some ways, minerals better mimic the activity of biological enzymes than free metal ions because some minerals can present surfaces that can bind reactants with high affinity.
Potential prebiotic metallopeptide sequences may exist in modern proteins
The protein enzymes thought to be the most ancient— based on the combined analysis of sequence, structure, and shared metabolism—all depend on metal ion coordination for their activity (Goldman et al. 2012). Furthermore, the structural features and amino acid sequences that coordinate metal ions are often highly conserved. Peptides based on such metal-binding motifs often retain the ability to bind the metal center (Mulholland et al. 1998). Therefore, in a somewhat analogous way to the metalloenzyme pathway example depicted in Figure 2, a path from free metal ion to metalloprotein can be envisioned to pass through intermediate metallopeptide states. In this way, short peptide sequences that confer some type of selective advantage could be gradually built upon to give longer, more active folds. If such a pathway transpired, then it may be possible to identify these early, potentially prebiotic, peptide sequences within the sequences of modern proteins.
Eck and Dayhoff (1966) were the first to consider the possibility that modern metalloproteins could have arisen from repeated sequences of short peptides. These authors noted that a 55-amino acid redox-active protein from the bacterium Clostridium pasteurianum likely resulted from a duplication event. Further, it was found that the protein could be broken down into progressively smaller repetitive units, all the way down to a tetrapeptide (a peptide consisting of four amino acids) (Fig. 4). Although the full-length protein coordinates two Fe4S4 clusters, the tetrapeptide was not assumed to bind a metal ion. Instead, it was proposed that later mutations resulted in properly positioned cysteines for iron–sulfur coordination (Eck and Dayhoff 1966).Iron–sulfur cluster proteins are believed to be evolutionarily ancient and are fundamental to several physiological processes, including central metabolism and protein and DNA synthesis. Hence, further effort into deciphering the origins of iron–sulfur proteins is important. The three most common iron–sulfur clusters are mononuclear, Fe2S2, and Fe4S4 clusters. In all these clusters, each tetrahedral iron is coordinated by a cysteine side-chain and, in the case of Fe2S2 and Fe4S4 clusters, the iron is additionally coordinated by inorganic sulfides (Fig. 1). Even without the use of complex search algorithms, several iron–sulfur cluster motifs are easily identifiable, much more so than for non–cysteine ligated metal centers. For example, one class of Fe4S4 proteins coordinate their cluster, in part, through a Cysteine–X–X–Cysteine–X–X–Cysteine motif, where X is any a-amino acid. These proteins additionally contain a fourth cysteine ligand further away in the primary sequence that is necessary to coordinate the cluster. Similar motifs can be found for specific classes of mononuclear iron and Fe2S2 proteins, although iron–sulfur clusters can be coordinated to quite different sequences.
The peptides that gave rise to iron–sulfur proteins may have impacted the evolution of other protein folds, too, because early metallopeptides were probably quite promiscuous in coordinating metal ions. Modern proteins that coordinate metal ions other than Fe2+ (e.g. Ni2+ and Zn2+) with the same ligand, such as happens with the cysteinyl thiolates, are plentiful. A more modern attempt to infer prebiotic metallopeptide sequences was made by van der Gulik et al. (2009) who searched the protein data bank for short sequences that primarily consisted of prebiotic amino acids [in that study, glycine (Gly), alanine (Ala), aspartic acid (Asp), and valine (Val) were designated as prebiotic amino acids] and that coordinated at least one metal ion. The search produced two different metal ion-binding motifs of Asp–X–Asp–X–Asp and Asp–X–X–X–X–Asp–X–Asp. These two motifs are associated in biology with the binding of Mg2+, Mn2+, Zn2+, and Ni2+.
Sequence analysis alone is not adequate to determine the prebiotic plausibility of metallopeptides. It is important to synthesize and test short peptide sequences to see if correlations can be drawn with the activity associated with more complex metalloprotein folds. Although still longer than what many would consider to be prebiotically plausible, a sixteen amino acid peptide taken from the sequence of an iron–sulfur protein from bacteria Peptococcus aerogenes ferredoxin was found to stably coordinate an iron–sulfur cluster in aqueous solution (Mulholland et al. 1998). More interestingly in terms of prebiotic relevance, the three amino acid peptide glutathione was shown to coordinate an iron–sulfur cluster (Qi et al. 2012). Each glutathione peptide contains one cysteine ligand, and so four glutathiones are needed to coordinate a single iron–sulfur cluster (Fig. 5). The sequence of glutathione is also attractive because glutathione contains a side-chain peptide bond that cannot be formed by the ribosome. If peptides existed before the ribosome, then the peptides would have likely contained a more heterogeneous distribution of backbone connectivity than that observed in modern proteins. Further, duplications of the sequence of glutathione gives a spacing of cysteine ligands similar to modern day ferredoxins (Scintilla et al. 2016). Of course, it is important to recognize that glutathione itself may not have existed on prebiotic Earth.
These insights show that it is important to dig deeper into “sequence space” by synthesizing and testing the iron–sulfur coordinating ability of a series of short peptide sequences so as to better understand the likelihood of an iron–sulfur peptide existing on prebiotic Earth. However, just existing is not enough. Unless metallopeptides can be shown to do something useful, their existence is meaningless. It would seem that identifying such activity should not be difficult: free metal ions themselves can catalyze reactions, and dipeptides bind divalent cations selectively (Belmonte et al. 2016). Non–metal ion coordinating dipeptides have also been shown to catalyze a few reactions, including peptide bond formation (Gorlero et al. 2009). It seems that the time is right to begin investigating how short peptides can enhance the intrinsic activity of metal centers.
Life as we know it depends heavily on metal ions to catalyze metabolic reactions, to transduce signals, and to form concentration gradients used to drive unfavorable reactions. Although most of contemporary metabolism is controlled by biological enzymes, non enzymatic metabolic reactions persist, including those catalyzed by metal ions. Further, at elevated temperatures metal ions themselves can catalyze the majority of the reactions of at least two fundamental metabolic pathways. Therefore, it is possible that of the many potential reaction networks that could have emerged, modern day metabolism reflects, at least in part, those reactions that were accelerated by metal ions. If peptides capable of coordinating the available metal ions were present on the early Earth then the reactivity of the metal center would have been modified in a sequence-dependent manner. Unfortunately, with the available data, we cannot conclude if a path similar to what has been described herein occurred. It is not even clear how such metabolic-like reactions could have been harnessed by protocellular structures. It is, therefore, important to experimentally explore the metal ion binding ability and catalytic activity of model prebiotic peptides to gain insight into what was and was not possible.
We thank C. Bonfio, S. Scintilla, and O. D. Toparlak for helpful comments on this manuscript. We thank the Simons Foundation (290358), the Armenise-Harvard Foundation, and COST Action CM1304 for financial support.
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