Structural Insight into [NiFe] Hydrogenase Maturation by Transient Complexes between Hyp Proteins

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• [NiFe] hydrogenases are crucial enzymes for reversible hydrogen production and consumption.
• Their active site contains a complex NiFe(CN)2CO cluster, requiring intricate maturation.
• Six Hyp proteins (HypABCDEF) orchestrate the biosynthesis and assembly of this active site.

Purpose of the Study:

• To elucidate the molecular mechanisms of [NiFe] hydrogenase maturation through structural biology.
• To provide atomic-level insights into the roles of Hyp proteins in cofactor assembly and insertion.

Main Methods:

• X-ray crystallography was employed to determine the structures of individual Hyp proteins and their complexes.
• Structures of transient complexes, including HypA-HypB, HypC-HypD, and HypA-HyhL, were resolved.

Main Results:

• Detailed molecular structures of all six Hyp proteins from *Thermococcus kodakarensis* were obtained.
• Structures revealed the mechanisms of nickel acquisition (HypA-HypB) and Fe(CN)2CO moiety assembly (HypC-HypD-HypE).
• The structure of the HypA-HyhL complex identified key checkpoints for maturation completion.

Conclusions:

• Structural data provide unprecedented mechanistic insights into [NiFe] hydrogenase maturation.
• The Hyp protein machinery facilitates sequential and regulated assembly of the NiFe(CN)2CO active site.
• This work lays the foundation for understanding and engineering hydrogenase enzymes.