[NiFe]-Hydrogenase Cofactor Biosynthesis and the Hyp Maturation Proteins (HypA-F) in E. coli

What the review examines

This minireview (Sawers, Hardelt, Haase and Lubek, Metallomics 2025) synthesizes recent biochemical and structural advances in how bacteria - with Escherichia coli as the principal model - biosynthesize and assemble the heterobimetallic cofactor at the heart of [NiFe]-hydrogenases. These enzymes catalyze the reversible interconversion of molecular hydrogen (H2 <-> 2H+ + 2e-) and are central to anaerobic energy metabolism in many bacteria and archaea.

The catalytic subunit of a [NiFe]-hydrogenase holds an unusual active site in which a nickel ion and an iron ion are bridged and coordinated by four cysteine thiolates. The iron additionally carries three diatomic ligands that are exceptional in biology: two cyanide (CN-) groups and one carbon monoxide (CO) group. These non-protein ligands hold the iron in a low-spin ferrous state and tune its redox chemistry so the site can activate hydrogen. Because the cell must synthesize toxic CN- and CO in a controlled, protein-templated way and install two different metals in the correct order, maturation requires a dedicated set of accessory proteins rather than spontaneous self-assembly.

The Hyp maturation proteins: division of labor

Six conserved accessory proteins, encoded by the hyp operon (hypA, hypB, hypC, hypD, hypE, hypF), carry out cofactor construction, assisted in E. coli by hydrogenase-specific chaperones such as HybG (a HypC paralog for hydrogenase-2) and HycI-type endopeptidases. The Hyp proteins fall into two functional groups that act on the two metals largely independently before the pieces are joined.

One group - HypC, HypD, HypE and HypF - is responsible for making the iron site and its CN/CO ligands. The second group - HypA and HypB - handles nickel: they bind, traffic and insert the nickel ion. This modularity means the iron subsite is fully built and delivered before nickel arrives, which the review highlights as a key control point that prevents mis-assembly.

Building the Fe(CN)2CO subsite: cyanide, carbon monoxide, and the scaffold

The two cyanide ligands originate from carbamoyl phosphate, a common metabolic intermediate. HypF acts as a carbamoyltransferase: it activates carbamoyl phosphate to carbamoyl-AMP and transfers the carbamoyl group onto the C-terminal cysteine of HypE, forming a thiocarboxamide. HypE, an ATP-dependent dehydratase, then dehydrates this group to yield a protein-bound thiocyanate - effectively a chemically caged cyanide handed off from the HypE cysteine.

Assembly of the iron unit occurs on a scaffold complex formed by HypD and HypC (or its paralog HybG). HypD is a redox-active protein that carries a [4Fe-4S] cluster and provides the surface on which iron is coordinated and the CN- ligands from HypE are attached; the small HypC/HybG chaperone binds and stabilizes the nascent Fe(CN)2CO unit and later carries it to the target hydrogenase subunit. Native mass spectrometry has identified the HybG chaperone as the physical carrier of the completed Fe(CN)2CO group. The review notes that the source of iron for this subsite is the Isc iron-sulfur cluster machinery, while the metabolic origin of the CO ligand under strictly anaerobic conditions remains an open question, with current evidence pointing toward one-carbon (C1) metabolism.

Nickel insertion comes last

Once the Fe(CN)2CO subsite is complete, HypC/HybG delivers it to the apo-form of the hydrogenase large subunit, and only then is nickel inserted. Nickel delivery depends on HypA and HypB working as a pair: HypB is a GTPase that provides a nucleotide-dependent switch for metal handoff, and HypA (a nickel- and zinc-binding protein) helps deliver the nickel ion to the iron-containing precursor. Insertion is coupled to GTP hydrolysis, giving the cell an energetic checkpoint over metalation.

A recurring theme the review emphasizes is metal specificity: the maturation machinery must select nickel and exclude chemically competitive ions such as zinc, which would otherwise mis-metalate the site and inactivate the enzyme. After nickel is in place, a dedicated protease cleaves a C-terminal extension of the large subunit, triggering a conformational change that internalizes and locks in the completed NiFe(CN)2CO center. This irreversible proteolytic step is the final commitment of a mature, catalytically competent hydrogenase.

Relevance to the metal-microbiome-disease axis

[NiFe]-hydrogenase maturation is a concrete example of how a trace metal - here nickel, alongside iron - is marshalled into a metalloenzyme, and it illustrates why nickel homeostasis matters for the microbiome. Enteric bacteria including E. coli, Salmonella enterica and the gastric pathogen Helicobacter pylori use [NiFe]-hydrogenases to extract energy from molecular hydrogen generated by the fermenting gut community. This hydrogen 'economy' is a shared currency of the microbiome, and the enzymes that tap it are entirely dependent on successful nickel loading by the Hyp system.

That dependence has disease consequences. Respiratory hydrogen use by Salmonella Typhimurium is required for full virulence, and in H. pylori both urease and [NiFe]-hydrogenase are nickel enzymes essential for colonizing the stomach. Because nickel availability gates these enzymes, host and environmental nickel status can shift which microbes thrive and how they colonize - a mechanistic link between metal exposure, microbiome composition and infection. It also frames a therapeutic angle consistent with nutritional immunity: restricting nickel, or disrupting Hyp-mediated maturation, can selectively disarm hydrogen-dependent pathogens. Conversely, altered environmental nickel loads (a heavy-metal exposure relevant to this site's editorial focus) could in principle reshape hydrogen-cycling members of the microbiome, though direct evidence linking exogenous nickel exposure to microbiome-mediated disease via hydrogenases specifically remains an area for further study rather than a settled result.