A Non-Covalent [4Fe-4S]/[2Fe] Interface in HydF Guides [FeFe]-Hydrogenase Maturation.

Area of Science:

• Biochemistry
• Bioinorganic Chemistry
• Enzymology

Background:

• Maturation of [FeFe]-hydrogenase requires the HydF maturase, a scaffold protein containing a [4Fe-4S] cluster.
• The precise role of the HydF [4Fe-4S] cluster and its interaction with the [2Fe] cofactor in H-cluster biosynthesis is not fully understood.
• Previous studies present conflicting data on cyanide linkage isomerism and the function of the cubane within the H-cluster.

Purpose of the Study:

• To elucidate the structural and mechanistic details of the HydF maturase's function in [FeFe]-hydrogenase maturation.
• To clarify the interaction between the HydF [4Fe-4S] cluster and the [2Fe] cofactor during H-cluster assembly.
• To reconcile conflicting data from previous studies on HydF function.

Main Methods:

• Utilized 57Fe nuclear resonance vibrational spectroscopy (NRVS) with selective isotopic labeling.
• Employed complementary protein structure predictions.
• Integrated data with prior spectroscopic, mutagenesis, and structural studies.

Main Results:

• Demonstrated that the [2Fe] subsite binds adjacent to the [4Fe-4S] cluster without a covalent cyanide bridge, maintaining electronic coupling.
• Protein structure predictions support this binding arrangement and reconcile prior experimental findings.
• Suggested the [4Fe-4S] cluster aids in assembling the CH2-NH-CH2 bridge of the [2Fe] site through interactions with the lipoate cofactor.

Conclusions:

• Provided a coherent structural and mechanistic framework for HydF function in H-cluster biosynthesis.
• Established a non-covalent interaction model between the [4Fe-4S] cluster and the [2Fe] subsite.
• Highlighted the role of HydF in positioning reactive components for efficient cofactor assembly.