Ligand Exchange in [2Fe–2S] Clusters

Replacement of Cysteines by Other Amino Acids

As for the [2Fe–2S]clusters, the first protein-bound clusters to be discoverd bearing non-cysteine ligands (Figure 1.3a)) were those found in Rieske proteins, originally dis-covered in 1964.^[59]While coordination by atoms other than sulphur was known from spectroscopic studies, the crystal structure of a water soluble fragment^[60] and subse-quently the whole cytochromebc₁complex^[61]of bovine heart mitochondria confirmed one iron atom to be bound to two histidine imidazole rings. This and other structures of Rieske proteins reveiled the[2Fe–2S]cluster to lie on the surface of the protein and thus to be solvent-exposed (Figure 1.3d)).^[62]

Figure 1.3: Naturally occurring[2Fe–2S]cluster variants with coordination by different amino acids.

— Details of the crystal structures of a) ferredoxin (PDB code 1QT9)^[63], b) biotin synthase (PDB code 1R30)^[64], c) mitoNEET (PDB code 2QH7)^{[65, 66]}, d) Rieske protein (PDB code 1RIE)^[60].

magnetically coupled (S=9/2) – in surprising contrast to other[2Fe–2S]clusters with antiferromagnetic coupling.^{[69, 70]}Similar mutant proteins could later be crystallised.^[71] Probably the most unexpected natural variant was reported in 2004 when the crystal structure of biotin synthase reveiled a cluster coordinated by an arginine residue (Fig-ure 1.3b)).^[64] However, both the exact coordination mode and the role of this highly exceptional ligand^[72]remained elusive.

In two crystallographically characterised proteins an exchange of just one cysteine by a histidine was found (Figure 1.3c)). The first is an outer mitochondrial membrane protein (called mitoNEET for its location in the mitochondria and the presence of the amino acid sequence Asn-Glu-Glu-Thr, NEET), crystallised independently by two groups.^{[65, 66, 73]}The redox-active [2Fe–2S] cluster can be removed under acidic condi-tions, probably due to protonation of the histidine residue.^[65]Obviously, mitoNEET is distinct from other known Fe/S proteins as no structural homologues were found, and all four ligands are contained within a small modular unit of only 17 amino acid residues.

However, this binding motif was then identified in seven distinct groups of proteins which are therefore also likely to be identified as yet unknown Fe/S proteins.^[73]With its function still unknown, mitoNEET – originally identified as a binding target of an antidiabetic drug – is of special interest since it is the first identified outer mitochondrial membrane protein containing an Fe/S cluster.

The other example of a natural coordination by three cysteines and one histidine was found in a protein involved in Fe/S cluster assembly, namely IscU which was crystallised as a homotrimer binding only one[2Fe–2S]cluster.^[74]Like in mitoNEET, the histidine is solvent-accessible.

1.3 Modulating Properties of Biological Fe/S Clusters

Replacement of Protein-Bound Cysteines by Smaller Molecules

Recently, two[2Fe–2S]clusters coordinated by ligands other than amino acid residues from the protein scaffold have been reported. In the first case, coordination is still pro-vided by cysteine residues but not from the protein environment: In a crystal structure of a poplar glutaredoxin C1 tetramer the present[2Fe–2S]cluster was found to be coor-dinated by two cysteine residues of different protein monomers and by two glutathione cysteines, one on each iron atom (Figure 1.4a)).^[75] Glutathione, which usually acts as antioxidant and as a cysteine reservoir, was found to be important for Fe/S cluster mat-uration in yeast.^[49]In fact, structural evidence for delivery of an intact[2Fe–2S]cluster – in a similar coordination environment – by a monothiol glutaredoxin was reported very recently.^[76]

An even more exotic coordination was found in the [FeFe]-hydrogenase maturase HydE where one cysteine residue is replaced by a water molecule (Figure 1.4b)).^[77] Together with HydF and HydG, HydE is crucial for the maturation of the active site of[FeFe]-hydrogenase and thus – like the glutaredoxins mentioned above – involved in the assembly of Fe/S proteins.

Figure 1.4: Naturally occurring[2Fe–2S]cluster variants with coordination by small molecules.

— Details of the crystal structures of a) glutaredoxin C1 tetramer with two glutathion molecules bound (PDB code 2E7P)^[75], b)[FeFe]-hydrogenase maturase HydE with a wa-ter molecule bound (PDB code 3CIX)^[77].

Minor Variations

Even minor variations less obvious than ligand exchange can have significant influ-ences on cluster properties. A prominent feature is the proton-coupled electron transfer

potential (+100 to+750 mV), the low-potential (−150 to+5 mV) and the intermediate-potential proteins (+60 to+100 mV).^[86]Apart from their different potentials, they can also be distinguished by the strong pH-dependence of the reduction potentials of the high-potential proteins in contrast to the relatively pH-independent potentials of the low-potential proteins. Furthermore, only the former and the intermediate-potential proteins^[87] contain a disulphide bridge close to the[2Fe–2S]cluster. While this disul-phide bridge is crucial for protein stability, it has only a minor influence on the redox potential.

Scheme 1.1: Model reaction for the proton-coupled electron transport in Rieske proteins.

The other biologically relevant amino acid ligand allowing for interactions different from those offered by cysteine is arginine as found in biotin synthase.^[64] In contrast to cysteine, this arginine residue is a possible bidentate ligand coordinating one or two metal atoms in a bridging fashion. Furthermore, both NH or NH₂groups and the N^εH group can act as hydrogen bond donors or acceptors, depending on the protonation state. Yet quite unlike the situation in the Rieske proteins, the role and even the exact coordination mode of this arginine are completely elusive to date. In a first approach, mutation experiments were performed which showed that this arginine ligand is not essential for the catalytic reaction which is described in detail in Chapter 1.4.^[88] It has been proposed that it may play electronic, mechanistic or structural roles, possibly

re-1.3 Modulating Properties of Biological Fe/S Clusters

lated to its bidentate nature or its positive charge in the protonated state.

Another example of secondary interactions was found in a [2Fe–2S] ferredoxin in which substantial conformational changes occurred upon reduction.^[89] First, the clus-ter itself was distorted from planar to a distorted lozenge. Second, a methionine side chain in close proximity was twisted by 180° bringing its sulphur atom within hydro-gen bonding distance of one of the sulphide bridges (Scheme 1.2). Although possibly connected with controlling the reduction potential of the cluster, its significance could not be explained yet.

Scheme 1.2: Structural changes upon reduction of Rhodobacter capsulatus [2Fe–2S] ferre-doxin.^[89]