Prof. Shiro Akabori, a pioneer of Japanese protein chemistry (former President of Osaka University and the first Director of the Institute for Protein Research), proposed the "surface metabolism theory." He suggested that life originated from chemical reactions (metabolism) occurring on the surface of pyrite (FeS_2) around deep-sea hydrothermal vents. This theory remains strongly supported today.
While the veracity of this theory has not been fully proven, the combination of iron and sulfur undeniably generates high reactivity, and it is a fact that modern organisms skillfully utilize these elements. Proteins containing cofactors known as Fe-S clusters are ubiquitous in the biological world and play essential roles in universal metabolic processes such as respiration and photosynthesis.
Although the structure of Fe-S clusters is simple, their assembly within cells is mediated by complex, multicomponent machinery. A representative example, the SUF machinery, consists of six enzymes/proteins (SufABCDSE) encoded by a specific operon.




　　　　　　
General Redox States

Multicomponent enzyme machinery involved in iron-sulfur cluster biosynthesis

[2Fe-2S] Cluster

• 2Fe^{3+}

  [Oxidized form]

• Fe^{3+} + Fe^{2+}

  [Reduced form]

[4Fe-4S] Cluster

• 2Fe^{3+} + 2Fe^{2+}

  [Oxidized form]

• Fe^{3+} + 3Fe^{2+}

  [Reduced form]

At the time I initiated my research in 2003, the specific roles of these protein groups were unclear. However, biochemical research on each component has progressed rapidly in recent years.
The SufS/SufE complex functions as a cysteine desulfurase (sulfur donor), and the SufA dimer is predicted to act as the iron donor. While the specific roles of SufB/SufC/SufD remain unknown, there is no doubt that they play a central function in the Fe-S cluster formation reaction. Interestingly, SufBCD forms a complex in which SufC functions as an ATPase. Since SufC shows sequence similarity (approx. 26%) to ABC-ATPases (the ATPase domains of ABC transporters, which serve as the power source for structural changes), we hypothesize that the molecular function of the SufBCD complex involves dynamic structural changes.
The purpose of this research is to elucidate the reaction and regulatory mechanisms of the SUF machinery: specifically, how each multi-component complex synthesizes Fe-S clusters and by what mechanism they transfer these clusters to apoproteins.

The SufC monomer is an inactive ATPase with a novel activity regulation mechanism
The primary structure of SufC shows similarity (20–30%) to the ATPase domain of ABC transporters (ABC-ATPase), which are membrane transport proteins. Sequences known as functional motifs of ABC-ATPases—such as Walker A, Walker B, and the ABC signature—are highly conserved in SufC. Indeed, SufC possesses ATPase activity, albeit weak.
In this study, we determined the crystal structure of Escherichia coli-derived SufC at 2.5 Å resolution. While the overall structure of SufC was similar to that of ABC-ATPases, the side chain of a conserved amino acid residue (Glu171) involved in ATP hydrolysis was oriented away from the nucleotide-binding pocket, forming a salt bridge with a side chain from another domain. As no such structure has been reported in ATPases to date, this finding revealed that SufC possesses a unique activity regulation mechanism.
(Please also refer to the Structural and Functional Analysis of the SufC2-SufD2 Complex)



　　　　　

Structure and Functional Analysis of SufD with a Novel Folding Motif
The function of SufD was entirely unknown due to the lack of sequence homology with other proteins. Therefore, to obtain clues regarding its function, we determined the structure of the SufD dimer.
The SufD structure exhibits a unique fold known as a $\beta$-helix and was identified as a novel structural protein classified into a new superfamily in structural motif databases. As it was difficult to predict the function from the three-dimensional structure alone, we decided to investigate residues involved in the function using genetic methods, bearing this unique structure in mind.

　　　　　　　　　　　　　　　　　
Crystal Structure of the SufD Dimer: Featuring a novel $\beta$-helix structure.
SufD Domain Structure: The dimer is formed by antiparallel $\beta$-sheets.
To investigate the structure-function relationship of SufD, we introduced mutations into amino acid residues at various sites and evaluated the in vivo function of these mutant SufDs using a complementation assay with E. coli mutant strains. This experimental system utilizes the exchange of temperature-sensitive plasmids; if SufD is non-functional, the cells cannot grow at 43°C.
The results were as follows:

1. Deletion of the N-terminal $\alpha$-helical domain had almost no effect on SufD function.
2. Deletion of the C-terminal $\alpha$-helical domain resulted in severe effects, and complementation ability was completely lost upon deletion of $\alpha$7–9.
3. Substitution of H360 at the dimer interface with any of the other 19 amino acids resulted in loss of function.

We experimentally verified that protein stability and complex formation ability were unaltered in the H360 mutant, strongly suggesting a direct involvement in Fe-S cluster synthesis. Since histidine can serve as a ligand for Fe-S clusters, H360 is expected to be a binding site for the nascent cluster. Interestingly, the side chain of H360 is completely buried within the molecule at the dimer interface (inside the $\beta$-helix).
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
In vivo Complementation Experiment:
Introduction of a mutation at H360 in SufD renders E. coli unable to grow.
Structure of the SufC{2}-SufD{2} Complex and Structural Changes in the ATPase SufC
Upon determining the structure of the SufC${2}-SufD${2} complex, we found that SufC adopts a conformation competent for ATP hydrolysis. As mentioned previously, SufC is inactive in its monomeric state; thus, it became clear that it is activated upon complex formation.
Furthermore, the two SufC molecules bound to the SufD dimer were positioned apart from each other within the complex. Based on the analogy with ABC transporters, it is hypothesized that the two SufC molecules form a dimer.
Therefore, we introduced cysteine mutations at the predicted dimer interface of SufC to investigate whether the residues come within a distance capable of forming a disulfide bond. The results revealed that SufC within the complex forms a dimer in an ATP- and Mg$^{2+}$-dependent manner.
　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
We were able to demonstrate through cross-linking experiments that the two SufC molecules within the SufC_2-SufD_2 complex form a dimer . The formation of the SufC dimer is expected to induce large conformational changes in the complex.
Operating Model of the SUF Machinery Involving Dynamic Structural Changes
Our results to date have shed light on the mechanism of Fe-S cluster biosynthesis in the SUF machinery .
We propose an operating model suggesting that the mechanism of the SUF machinery serves as the prototype for the ABC transporter superfamily—the largest in the biological world. This points to the surprising possibility that completely different functions (cluster synthesis vs. transmembrane transport) are achieved through a common underlying principle . We are currently conducting research aimed at verifying this model.



It has emerged that conformational changes in the complex induced by ATP binding expose ligands buried within the molecule (some of which have already been identified), thereby facilitating the synthesis of Fe-S clusters.