Over 4 billion years ago, Earth was devoid not only of life, but the building blocks of life, called biomolecules — organic, carbon-based structures that react with one another, organizing into complex biochemical systems and ultimately, life. Where did these biomolecules come from?
The byproducts of biomolecular reactions offer investigators critical clues called biosignatures — chemical fingerprints hinting at the origins of life. University of Wisconsin-Madison PhD student Holly Rucker studies some of the oldest biosignatures currently known, left behind by ancient enzymes in three-billion-year-old rocks, to uncover, through computational analysis, the complexity of early life.
How did biomolecules form life?
Scientists think Earth was volatile and hostile to potential life for hundreds of millions of years after its formation. Geological evidence suggests frequent barrages of meteorites, molten rock and a lack of water. But this volatility likely supplied the energy for chemical reactions that synthesized the first “biologically available” molecules.
Biomolecules are reactive in the presence of heat, UV radiation from the sun and electrical energy from lightning — common forces on early Earth — driving the reaction of more complex structures.
Scientists believe life began over three billion years ago. The challenge remains in understanding how biomolecules were organized into life, and the answer lies in a key biomolecular player known as the peptide. Peptides are sequences of amino acids, a type of biomolecule. Distinct amino acid combinations confer structural and functional properties in peptides. After peptides came enzymes, complex combinations of amino acids that can speed up chemical reactions.
This organization of amino acids into peptides helps us understand how chemical reactions sped up from slow changes over hundreds of millions of years to a pace that could support the fast reactions required for life.
Astromicrobiology
Rucker studies ancient enzyme biosignatures under Dr. Betül Kaçar, who pioneered a field called molecular paleobiology. While traditional paleontology benefits from novel genetic tools, Rucker’s field couldn’t exist without them.
Rucker studies the sequences of enzymes, the molecular protein machines that powered the very first cells. Bones and plant matter fossilize. Enzymes do not, but they do leave behind signature biosignatures. With the right computational tools, ancient protein sequences can be reconstructed, and even brought back to life, to test what made its signature in ancient rocks.
What Is nitrogenase?
At the center of this story is a single enzyme called nitrogenase. Nitrogenase, Rucker said, catalyzes a reaction that is among the most important chemistry on Earth: converting atmospheric nitrogen into digestible energy.
“Nitrogen is involved in pretty much every single biological process,” Rucker said. “Your DNA, proteins, making your cells, everything — biology is nitrogen, in a sense.”
The atmosphere is roughly 78% nitrogen gas. There’s more of it than anything else up there. But atmospheric nitrogen is locked in its chemical structure, a nearly unbreakable triple bond that’s stable and, for a while, was biologically inaccessible.
Bacteria and archaea developed the ability to “fix” nitrogen — to crack that triple bond and convert atmospheric nitrogen into ammonia that living things can actually use — more than 3 billion years ago, before cells with a nucleus ever existed. Nitrogenase, the enzyme that does this job, is large and structurally complex, requiring enormous energy to run.
“It’s a very big, bulky, multi-subunit enzyme complex that has to come together for this catalysis to happen,” Rucker said.
Humans didn’t solve the same nitrogen problem until the early twentieth century when population growth was outpacing our ability to produce enough nitrogen-dependent fertilizers to support our agricultural needs. The Haber-Bosch process — developed by chemist Fritz Haber during World War I — can fix atmospheric nitrogen industrially, and it now accounts for roughly half of all fixed nitrogen on Earth.
The catch: the process now consumes 1-3% of the entire planet’s annual energy expenditure.
“It’s an insane amount of energy. Life figured out how to do the same thing billions of years ago,” Rucker said.
Nitrogenase research is important for our past and future. In response to agricultural nitrogen dependency, researchers are harnessing nitrogenases to engineer plants that can autonomously fix nitrogen.
Building a protein family tree
How do you reconstruct an enzyme that no longer exists? Rucker describes the approach as building a “protein family tree” — then reading it backwards.
First, Rucker compiled every known modern nitrogenase gene sequence using living bacteria and archaea. Then, the lab feeds those sequences into computational models that trace mutations and evolutionary pressures backward through the branches of the tree toward a common ancestor. At any node on that tree, the model yields a predicted ancestral sequence — the most statistically likely version of the protein at that moment in evolutionary history.
“We use a bunch of models that use computational techniques to estimate mutation and evolutionary forces, to predict how the protein sequence would change over time,” Rucker says.
Once the ancestral sequence is predicted, it’s synthesized and inserted into a modern bacterium that naturally performs nitrogen fixation. Deprived of external nitrogen sources, the ancient enzyme works if the bacterium grows.
The oldest enzymes recreated in this study share roughly 60-70% of their amino acid sequence with modern nitrogenase. Hundreds of millions of years of evolution have changed the protein substantially, but something essential has stayed the same.
The method has limits. Because the reconstruction is built from lineages that survived to the present, “it is somewhat a history written by the winners,” Rucker said. Proteins from extinct lineages — ones that left no living descendants — are simply invisible to the model.
And there’s more work to be done before Rucker’s enzymes can be used for modern-day nitrogen fixation. The enzymes Rucker created are adapted to Earth’s early oxygen-scarce environment, and nitrogenases are sensitive to our oxygen-rich environment. This is a current challenge for people studying this potential solution.
But Rucker said this doesn’t undermine the novelty of the work. “This paper is the first to make this many sequence-level changes to nitrogenase and measure [the downstream effects on its chemical output],” she said.
Burning ancient bacteria
Rucker measures a chemical signature called a nitrogen isotope ratio — specifically, the proportion of nitrogen-15, which carries an extra neutron, to nitrogen-14 inside the bacterial cells. The result is a value that acts as a chemical fingerprint. Different nitrogenases produce distinct fingerprints. Rucker’s ancient enzyme signatures match those found in ancient rocks.
“The [fingerprints] are distinct enough that there is no ambiguity between them,” Rucker said.
The technique for measuring the fingerprints is called isotope ratio mass spectrometry. In order to prepare samples, Rucker burns extra-concentrated bacteria samples.
“We grow our cultures of bacteria that have the ancestral enzyme,” she said. “We take subsamples, we get a pellet, and then we burn it — dry it out, wrap it up in basically foil, and in the instrument it drops down, combusts it, turns it all into a gas, and then measures the nitrogen isotope of the entire cell.”
Because the bacteria are given no external nitrogen source, every nitrogen atom inside them arrives from the enzyme. The isotope ratio of the whole cell reflects the work of the ancient nitrogenase.
Ancient doesn’t mean simple
Rucker said nitrogenase defies the modern conception “that early life was simple, and that evolution has somehow made life better — made enzymes better.”
Nitrogenase, a multi-subunit enzyme complex that breaks one of the most chemically stable molecules in nature, consuming sixteen units of cellular energy per reaction, is not a primitive system by any reasonable definition. Converting atmospheric nitrogen was “a very complex process that life figured out how to do almost immediately,” Rucker said. “It’s very often that people assume that when we look at these ancestors, the farther back we go, the worse they’re going to be,” she said.
Her data tells a different story. The ancient nitrogenases perform the same chemistry as their modern counterparts. Under some conditions, they outperform them. “I think that’s a very complex process that life figured out how to do almost immediately,” she said.
Complexity is a matter of perspective and scale, and there is plenty of it, even at the microbial level, and even 3 billion years ago.
Biosignatures and the search beyond Earth
Rucker’s work is funded in part by NASA, connecting directly to one of the agency’s core scientific interests: biosignatures that could be preserved across geological time and detected remotely, whether in an ancient rock, a planetary atmosphere, or a soil sample returned from another world. For a biosignature to be useful, it needs to be unambiguous — a sign of life stable enough to survive billions of years.
The nitrogen biosignature Rucker measured proved durable across enormous variations in enzyme sequence, protein structure, and environmental conditions, including the oxygen-free early Earth.
“If we were to go elsewhere looking for life and we’re looking for signs of nitrogen fixation, we’re going to be looking, most likely, for that signal,” she said. “It seems to be really robust to all of these different parameters.”
On Mars, any attempt to condition soil for plant growth would require nitrogen fixers, and that same biosignature could, in principle, be what a future mission would look for as evidence that life had already been there. Rucker’s work doesn’t prove life exists elsewhere, but it gives scientists a more precise instrument for asking the question.
Astrobiologist by day, comedian by night: Two sides of Holly Rucker
Rucker’s path to astrobiology started with science fiction. “I learned about astrobiology through watching sci-fi movies,” she said. “I was like, what’s the actual science behind all of these movies? And I found astrobiology.”
She studied biology at James Madison University in Virginia, then made a deliberate pivot — a master’s in marine and atmospheric science at Stony Brook University, with a focus on marine geochemistry — to make herself a stronger candidate for the Kacar Lab.
“I knew that was going to be the lab I wanted to work in, so I worked really hard,” she said. Rucker joined in 2022, and the ancestral enzyme project became her primary focus.
When Rucker isn’t growing ancient bacteria or running isotope calculations, she’s onstage at Atlas Improv in downtown Madison where she performs four shows a week, teaches improv classes and coaches independent teams. She also performs science-based comedy shows with a prepared slideshow, though she improvises the rest.
She didn’t always see herself here. In high school she toured art colleges and she is the first scientist in her family. Her mother has a college degree, while her father does not. None of this was the obvious path.
Inspired by art that questions the wildest possibilities, Holly chose research, following evidence toward what she calls “better-informed what-ifs.”
