NiFe(CN)2CO Cofactor Biosynthesis in E. coli: Review

Overview

This minireview synthesizes recent advances in how bacteria and archaea assemble the heterobimetallic NiFe(CN)₂CO cofactor of [NiFe]-hydrogenases, emphasizing ligand origins, Hyp protein functions, scaffold-mediated Fe(CN)₂CO assembly, and ATP/NTP-coupled nickel insertion with clinical relevance for redox metabolism and gas-handling pathogens.

What was studied and how?

This is a minireview focusing on the biosynthesis of the [NiFe]-hydrogenase cofactor and its assembly into the large subunit active site. The primary “matrix” is the intracellular maturation machinery (Hyp proteins and ancillary factors) that builds and inserts Fe(CN)₂CO and Ni into apo–large subunits across diverse microbes, notably Escherichia coli, Cupriavidus necator (Ralstonia eutropha), Thermococcus kodakarensis, and Rhizobium leguminosarum. Metals/metalloids are Ni and Fe; speciation is explicitly NiFe(CN)₂CO with Fe bound to two CN⁻ and one CO; a transient Fe(I)–CO intermediate is proposed, with Fe likely introduced initially as Fe²⁺. Methods summarized include IR spectroscopy identifying intrinsic CO and CN ligands; X-ray crystallography of Hyp proteins and proteases; native mass spectrometry showing HypC/HybG shuttling of Fe(CN)₂CO; UV–visible spectroscopy of [4Fe–4S]-HypD; ATPase/GTPase assays of HypCD and HypB; and AlphaFold3 structural simulations for HypCD and a predicted HypCD–AccB interaction. Instrument models/settings not reported. The diagram on page 6 summarizes the four-step pathway: Fe(CN)₂CO synthesis on HypCD, transfer to apo–large subunit via HypC/HybG, Ni insertion by HypAB/HypB, and C-terminal proteolysis sealing the site.

Most important findings

Critical point	Details
Cofactor identity and ligation	Active site cofactor is NiFe(CN)₂CO; Fe carries one CO and two CN⁻; four conserved cysteines (±selenocysteine) coordinate Ni and bridge to Fe.
Cyanide origin	CN⁻ derives from carbamoyl phosphate via HypF-HypE: carbamoylation of HypE C-terminal Cys, ATP-dependent dehydration to a thiocyanate, and reductive transfer to Fe on HypCD.
Carbonyl origin	Under oxic conditions, HypX generates CO from N¹⁰-formyl-THF via a formyl-CoA intermediate; the anoxic CO source remains unresolved, with candidates in C₁ metabolism (formyl intermediates/formate/CO₂).
Scaffolded Fe(CN)₂CO synthesis	Fe(CN)₂CO assembles on the HypC–HypD scaffold; HypD is a [4Fe–4S] protein with conserved cysteines and a Cys69/Cys72 redox motif implicated in CN⁻ transfer; ISC machinery likely supplies Fe.
Transfer to apo–large subunit	HypC/HybG binds apo–large subunits and not mature ones; ATP binding to HypCD promotes release/transfer of Fe(CN)₂CO without requiring hydrolysis in vitro.
Nickel delivery	HypA/HypB (GTPase/ATPase variants exist) deliver Ni specifically; nucleotide-bound HypB induces a high-affinity Ni site on HypA; Ni insertion precedes C-terminal proteolytic processing.
Proteolytic checkpoint	Endoproteases (HycI/HybD family) cleave a C-terminal peptide only when Ni is correctly installed, closing the active site; some hydrogenases lack this peptide yet mature by alternative means.
O₂-tolerant systems	Ancillary proteins (HoxV/HupK and HoxL/HupF paralogues) appear to protect or transiently store Fe(CN)₂CO and facilitate maturation under O₂; HypX links CO supply to C₁ metabolism.
Structural predictions	AlphaFold3 suggests a plausible HypCD–AccB (biotin carboxyl carrier) interaction positioning biotinyl-CO₂ near the Fe site, consistent with a speculative ATP-driven dehydration model.

Strengths

The review integrates genetics, spectroscopy, structural biology, and biochemistry into a coherent maturation pathway spanning Fe(CN)₂CO synthesis, transfer, Ni insertion, and proteolytic sealing. It is source-faithful to primary mechanistic evidence, highlights redox-active motifs in HypD, and links Fe sourcing to ISC biogenesis, increasing plausibility for scaffolded Fe delivery. The coverage of HypX as a defined CO-generating enzyme in oxic maturation and of ancillary O₂-tolerance factors strengthens translational relevance where pathogens encounter oxygen gradients. Use of native mass spectrometry and AlphaFold3 enriches mechanistic hypotheses and identifies tractable interfaces for experimental validation.

Any Limitations

As a minireview, it provides no new quantitative concentration data, clinical matrices, or method validation. The anoxic CO source remains unresolved, and electron donors to HypD’s [4Fe–4S] cluster are undefined. Some proposals (e.g., HypCD–AccB coupling and ATP-driven CO₂ dehydration) are based on predictions or indirect observations and need in vivo corroboration.

Future Perspectives

Clinically relevant organisms that rely on [NiFe]-hydrogenases could be targeted by inhibiting specific maturation steps: HypF–HypE carbamoylation/dehydration (cyanide delivery), HypCD ATP-dependent transfer, HypAB/HypB nucleotide cycles (Ni fidelity), or O₂-tolerance modules (HoxV/HupK/HoxL/HupF, HypX). Resolving the anoxic CO pathway and HypD electron donors would unlock diagnostic probes (e.g., ligand-state–specific IR signatures) and therapeutic strategies disrupting cofactor assembly under host conditions. Structural capture of Fe(CN)₂CO-loaded HypCD and ternary transfer states should guide inhibitor design.

Conclusion

This review consolidates a four-stage, scaffold-centric model for [NiFe]-cofactor biogenesis: CP-derived cyanide synthesis on HypE/F, Fe(CN)₂CO assembly on HypCD, ATP/NTP-coupled Ni delivery by HypA/HypB, and protease-gated active-site closure, with HypX and O₂-tolerance factors modulating CO supply and maturation in air. Outstanding questions—chiefly the anoxic CO source and HypD electron input—define the next experimental frontier with clear translational targets in microbial metallomics.

Citation

Sawers RG, Hardelt M, Haase A, Lubek D. Biosynthesis and assembly of hydrogenase [NiFe]-cofactor: recent advances and perspectives. Metallomics. 2025;17:mfaf015. doi:10.1093/mtomcs/mfaf015