Exploring the structural diversity of large hydrogenase fusion proteins in eukaryotic anaerobes

Abstract

Hydrogen is essen/al for sustaining microbial communi/es in a wide range of modern ecosystems and was likely pivotal for the emergence of early life on our planet. Across the prokaryotes and eukaryotes, hydrogen is produced or consumed by ‘hydrogenase’ enzymes. Hydrogenases in prokaryotes vary in their subunit composi/on, ranging from single-subunit soluble proteins to large, mul/-subunit complexes. Varia/ons in this subunit composi/on impact the overall reac/on of the complex whereby hydrogen produc/on is coupled to other ac/vi/es related to maintaining the cell’s redox balance by, for instance, oxida/ng/reducing NADH or ferredoxin. Most eukaryo/c hydrogenases are single-subunit enzymes that catalyse the reduc/on of protons using electrons derived from ferredoxin proteins, ul/mately producing hydrogen gas. However, some eukaryotes encode hydrogenase proteins fused with addi/onal domains, implying they might possess alterna/ve ac/vi/es. To explore the diversity of hydrogenase proteins in eukaryotes, we focused on four fusion proteins iden/fied in the transcriptomes of Nyctotherus ovalis (ciliate), Sulcionema specki (diplonemid), and Pygsuia biforma (breviate). Using protein folding methods including AlphaFold 3, Chai-1 and Boltz-1, we found that the eukaryo/c fusion proteins are predicted to form complexes with similar structural features as homologous prokaryo/c mul/-subunit complexes. We aTempted to heterologously express these proteins in different strains of E. coli but faced challenges due to their complex nature, so we focused the work on purifying the S. specki protein from inclusion bodies using different denaturing agents.

The characterisa/on of these eukaryo/c proteins that likely have unique hydrogen-associated ac/vi/es aids in our understanding of how pro/sts adapt to anaerobic environments.

Popular Abstract

Hydrogenases: ancient enzymes in a modern world

Life as we know it began in the absence of oxygen. Long before other organisms filled our atmosphere with this gas, ancient microbes likely relied on hydrogen (H2) to survive. But H2 is far from a thing of the past, it still supports millions of microorganisms in nearly every environment on Earth. More recently, H2 has also emerged as a promising alternative for green energy production.

There are many ways we could produce H2, but microbes unlocked this secret long ago. They developed a group of specialized enzymes – hydrogenases – that can both consume and produce this gas. These microscopic power houses, found across the whole tree of life, split H2 molecules intro protons and electrons (or the reverse), fuelling key processes in the absence of oxygen.

Among the types of hydrogenases, [FeFe]-hydrogenases are the most efficient at generating H2. They are incredibly diverse and can exist as single units (monomers) or as part of larger protein complexes. A few years ago, researchers discovered something unusual: in a single-celled eukaryote (a protist) some of these protein complexes – from bacterial origin – had fused into a single large enzyme. At the time, it was the only known case.

Now, in our lab, we have found more of these fusion proteins. This raises exciting questions: do these fused enzymes function differently than their bacterial ancestors? Do they share the same structure? Have they evolved new capabilities?

In this study, we combined bioinformatics and lab experiments to investigate four of these fusion proteins. We predicted their structures, suggested potential mechanisms to explain how they function, and attempted to produce them in bacteria. Despite some challenges, we managed to express all four and purified one of them. This work is the first attempt to characterize a group of enzymes that had previously been reported only once, with the hopes that it will help us better understand how eukaryotes have adapted to low-oxygen environments and how they produce hydrogen.

Master’s Degree Project in Molecular Biology 60 credits 2025
Department of Biology, Lund University


Advisors: Courtney Stairs and Humberto Itriago
Department of Biology, Lund University

• author: Gallego Rodriguez, Violeta
• supervisor: Courtney Stairs; Humberto Itriago
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20242
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9214494
• date added to LUP: 2025-10-24 13:58:23
• date last changed: 2025-10-24 13:58:23