At the dawn of life, did metabolism come first?

Four billion years ago, our planet was water and barren rock. Out of this, some mighty complicated chemistry bubbled up, perhaps in a pond or a deep ocean vent. Eventually, that chemistry got wrapped in membranes, a primitive cell developed and life emerged from the ooze.

But how? Among the many mysteries is a chicken-and-egg problem to solve. The proteins called enzymes that get chemical reactions going inside cells are created from instructions carried in genetic material: DNA or RNA. But at the start, those molecules weren’t around: To make them, you need enzymes.

So what got things going?

One idea long floated by scientists is that genetic material came first — in the form of a molecule called RNA, a close cousin to DNA. RNA’s beauty is its versatility: It can catalyze chemical reactions and store genetic information. So perhaps in a pond on Earth’s surface, molecules were concentrated by evaporation and then linked together to form the first RNA strands.

But so far, scientists have not been able to create RNA molecules in experiments mimicking the soups of simple chemicals that would have been around on early Earth. “There have been reports of how to do it, but they always seem a little bit contrived,” says Albert Fahrenbach, an organic chemist at the University of New South Wales in Sydney — though he adds that proponents say such trickiness is only to be expected.

It’s difficult to imagine that a system of self-replicating chains of RNA could have organized spontaneously, says Robert Pascal, a chemist who works on the origins and emergence of life at Aix-Marseille University in France. “I think that really, nobody believes now that it could have been possible.”

Another possibility is that biochemistry came first — it evolved as geochemistry, outside of cells. Chemical reactions would have proceeded without enzymes at the start, very slowly. Reactions would inch forward because they were thermodynamically favored, and possibly sped up by heat or metals. Later on, primitive enzymes developed, further speeding up that primordial chemistry of life.

Over geological time, geochemistry would have become faster and more elaborate, adding on new reactions. Somewhere along the way, cell membranes and a system of heredity, in the form of RNA or DNA, would have arisen. Geochemistry would morph into biochemistry.

This second hypothesis lacked key experimental evidence until fairly recently. But in the last few years, researchers have been able to test in the lab vast combinations of chemical mixtures and conditions and identified ways to replicate core metabolic reactions that take place in cells — all without enzymes.

The idea that geochemistry preceded biochemistry is “a really powerful idea,” says Susan Lang, a geochemist at Woods Hole Oceanographic Institution in Massachusetts. “And I think that [the scientists] have brought a lot of evidence to bear to support that idea.”

Inorganic origins

As far back as 1910, the Russian biologist Konstantin Sergejewitch Mereschkowsky reasoned that the very first cells had to produce organic molecules — the stuff of life — from inorganic substances. Specifically, they had to take hydrogen (H₂) and carbon dioxide (CO₂) to make organic molecules like fatty acids, sugars and amino acids.

About 20 years ago, evolutionary microbiologist Bill Martin at the University of Dusseldorf in Germany and geochemist Mike Russell at the NASA Jet Propulsion Laboratory in California proposed that life started in a location suitable for these critical reactions: deep-sea hydrothermal vents.

Within these vents, iron in rock reacts with water to produce hydrogen. And that hydrogen could react with CO₂ to produce simple organic molecules that are central to cell biochemistry: formate, with its one carbon atom, acetate with two carbons, and pyruvate with three carbons.

Guided by this idea, in 2010 Lang and her team at Woods Hole, working in the Lost City, a hydrothermal field in the middle of the Atlantic Ocean, confirmed that small organic molecules are indeed produced in vents quite separate from the activity of microbes that live in these extreme environments.

A microbial pathway

The scientists also noticed that steps in these geochemical reactions at the vents are identical to the way that microbial cells living near the vents make organic molecules from CO₂ and hydrogen. This fit nicely with the idea that biochemical pathways got their start as geochemical reactions, with enzymes evolving only later.

Indeed, this particular set of reactions, known as the acetyl-CoA pathway, is very ancient, Martin and colleagues have shown. It is shared by two fundamental groups of life, bacteria and archaea — and therefore traces all the way back to the last common ancestor of all life on Earth.

But today’s microbial cells need a cool 127 enzymes to make that three-carbon pyruvate. Could the scientists replicate this pathway in a lab, without enzymes, as would have been the case at the dawn of life? Martin, his then-student Martina Preiner, chemist Joseph Moran, now at the University of Ottawa, Canada, and their colleagues recently showed that indeed they could.

In one of the reports, published in 2020, Preiner conducted tests in a series of chemical reactors that could sustain high heat. Into these containers she put carbon dioxide, as well as vials holding water plus iron or nickel in various proportions. Then she left them to react overnight.

Preiner’s breakthrough came when she figured out she needed to run the reactors with one metal at a time and control the levels of hydrogen that were present. That done, the reactors reliably produced formate, acetate, pyruvate, methanol and methane — all of which are made by bacteria.

“These metals replace 127 enzymes and give us five products which are exactly the products of the biological pathway,” Martin says.

The scientists note that iron and nickel are both found in deep-sea vents where they could have helped to shepherd pre-biological reactions.

What’s more, these metals are still to be found in the parts of the modern-day enzymes that catalyze the acetyl CoA pathway inside cells. “It’s the metals that were there first, and then the enzymes incorporated the metals, but the metals remain the essential catalysts,” Martin says.

Metals were abundant back on the primordial planet. Iron, in particular, was everywhere, says Markus Ralser, a metabolism researcher at Charité – Universitätsmedizin Berlin. “You cannot avoid an iron chemistry, and that’s why we have it everywhere in the chemistry that’s implemented in our cells,” he says.

Advancing without enzymes

The pyruvate that Preiner, Martin and Moran made in their experiments is essential for another important part of cell metabolism: making amino acids and nucleotides. Living cells need amino acids to link together into proteins, and they need nucleotides to make DNA and RNA.

The core assembly line that makes these substances is called the reverse TCA cycle, and pyruvate is one of the key inputs into the cycle.

Moran has been working to replicate the reverse TCA cycle outside of cells for the better part of a decade. When he started, origin-of-life researchers had many great ideas, but few were having success doing experiments. He wanted to apply his expertise in chemical catalysis to important questions in the field.

The main challenge, as Moran saw it, was that although the reactions in the reverse TCA cycle are theoretically possible — meaning that chemical properties would ultimately push them forward, like gravity would favor a hike downhill — some of these reactions take an awful lot to get started. They have what’s called a high activation barrier, like a small hill to climb before that downhill hike can begin. Would it be possible to get over these activation barriers without enzymes? Under what conditions?

“The way I approached the problem was coming from the world of chemical catalysis development and screening, which is, ‘Let’s not think about it too hard, let’s design some experiments that can be run, where we can run a huge number of experiments in parallel, and we’ll just empirically search for it in a very efficient way,’” Moran says.

Starting in 2015, Moran and his team (then at the University of Strasbourg in France, where he still does some work) studied each of the 11 core reactions in the reverse TCA cycle, testing every metal and non-organic catalyst they could get their hands on, under every condition they could imagine: at different temperatures and pH values, in the presence of different metals and minerals.

Some test tubes would try to complete step one: the conversion of pyruvate to oxaloacetate. Another would aim to carry out the next reaction, from oxaloacetate to malate. And so on. In addition to the core reactions in the reverse TCA cycle, the scientists more recently investigated the reactions that branch off from this cycle to make nucleotides, amino acids and sugar phosphates.

The team used an automated mechanism to run samples 24 hours a day, day after day. “We just blasted through it for three years,” Moran says.

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In 2019, Moran’s team reported that they could make nine of the 11 reverse TCA cycle metabolites without enzymes. They also identified conditions in which six of the 11 reactions can all work together, in the same test tube. “The vast majority of the reactions and metabolism seemed easy enough that they can actually happen without enzymes,” Moran says. “At the same time, we have to be realistic and say that we have not identified any conditions that allow an entire metabolism to emerge; we find conditions that allow little stretches of it to happen here, and specific reactions there.”

But Moran is optimistic about the prospect of re-creating a non-enzymatic metabolism. It will just take some creativity to identify conditions under which some of the more tricky reactions, like the conversion of pyruvate to oxaloacetate, can occur, he says.

“What Joseph did in the last decade was really remarkable,” says Preiner, who now leads a lab group at the Max Planck Institute for Terrestrial Microbiology in Marburg, Germany. “That was one of the most important things that anyone has done in the last 50 years, for the origin of life — to really look at how certain reactions can work non-enzymatically."

Small but crucial missing pieces

As they continue their work on the start of life’s chemistry, Preiner and Moran have both turned to small chemicals called cofactors or coenzymes, which assist in enzyme reactions.

These cofactors are extremely important. One called NAD+ transfers electrons between molecules — a crucial job during reactions — and one called SAM takes and gives methyl groups to organic chemicals that are being processed. “They do quite simple tasks, but very, very, central and important tasks of metabolism,” Preiner says. It’s hard to imagine that they or some chemical like them wasn’t involved early on, she adds.

The scientists want to know what role these cofactors could have played at the dawn of life, and how they became integrated into geochemistry, then biochemistry.

Today’s cofactors, they are learning, can have catalytic activity like an enzyme does. And some can even catalyze their own production. It would therefore make sense, Moran says, that these cofactors would have been crucial for speeding up metabolic reactions. They could have accelerated some chemical pathways over others, helping to sculpt a biochemical network.

The scientists also think that some of those cofactors had a part to play in a pivotal step in cellular evolution: the emergence of genes. One cofactor called NADH, they note, consists of two nucleotides — the building blocks that RNA and DNA are made of.

“As I imagine it right now, it might be that these molecules, on an early Earth, were being over-produced. And so they got different tasks — parts of them got into RNA, and others went into metabolism,” Preiner says.

The researchers know that, of course, they can never know exactly how life came to be: They are like archaeologists or paleontologists trying to piece together the deep past from sparse, crumbled relics. Indeed, comments Pascal, there was nothing preordained about the metabolism of life that persisted and won out among many other possible self-catalyzing chemical systems. “This result is not the only one which is possible,” he says. “We have many other possibilities.”

Still, the scientists have shown that it is possible to create a non-enzymatic metabolism that is uncannily similar to that of microbes in the hydrothermal vents — providing key, concrete evidence for a century-old theoretical idea about life’s origins. Like fossils in ancient rocks or artifacts at old grave sites, the biochemical reactions inside living cells connect us to a time long before the most primitive life existed on the planet.