Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA

As a graduate student in Doug Rees' lab at Caltech, my studies centered on the X-ray crystal structure determination of a number of metallo-proteins (particularly those containing various forms of iron) from various microbes in order to better understand how these proteins function. The iron proteins studied were as follows:

Superoxide Reductase (SOR) from the hyperthermophile Pyrococcus furiosus

Superoxide reductase (SOR) is a non-heme mono-iron protein that functions in anaerobic microbes such as Pyrococcus furiosus as a defense mechanism against reactive oxygen species by catalyzing the reduction of superoxide to hydrogen peroxide. The crystal structures of SOR in both its oxidized and reduced states were determined to 1.7 Å and 2.0 Å resolution, respectively (PDB IDs 1do6, 1dqi, 1dqk). The structures revealed that the protein is a homo-tetramer, with each subunit adopting an immunoglobulin-like (Ig) fold and containing one catalytic active site iron per subunit (Figure 1a). The active site irons were found to exhibit octahedral coordination in the oxidized form of the structure and penta-coordination in the reduced form (Figure 1b). From these structures, we postulated a possible mechanism by which superoxide accessibility may be regulated. (Yeh et al, Biochem., 2000)

Figure 1. (a) Ribbons diagram representation of the homo-tetrameric arrangement of SOR. Subunits A, B, C, and D are depicted in yellow, green, blue, and red, respectively, while iron atoms are depicted as gold spheres. (b) Electrostatic potential map contoured from -15 kBT (red) to +15 kBT (blue) showing the predominantly uncharged and relatively exposed environment of the iron center active site in reduced SOR. Glu 14 and Lys 15 are the closest charged residues to the iron, depicted as a green sphere.

[2Fe-2S] Ferredoxin 4 (Fd4) from the hyperthermophile Aquifex aeolicus

Ferredoxins are iron-sulfur proteins that mediate electron transfer in a wide variety of metabolic reactions. The structure of the two-iron/two-sulfur [2Fe-2S] ferredoxin (Fd4) from Aquifex aeolicus, a thermophilic bacterium, was determined by X-ray crystallography to 2.3 Å and subsequently to 1.5 Å, and revealed a thioredoxin-like fold that is novel among iron-sulfur proteins (PDB IDs 1f37 and 1m2a). Protein sequence alignments showed that this fold is present as components of more complex anaerobic and aerobic electron transfer systems (e.g., complex I of aerobic respiratory chains). (Yeh et al., J. Mol. Biol., 2000)

Figure 2.  (a) Ribbons diagram representation of the homo-dimeric arrangement of Fd4. Subunits A (green) and B (purple) are shown with their respective [2Fe-2S] clusters represented by ball-and-stick models. Iron and sulfur atoms of the clusters are colored green and yellow, respectively. (b) The immediate environment around the [2Fe-2S] cluster. Potential hydrogen bonds between the cluster sulfide S1 atom and the amide nitrogen of Cys 22 and the guanidinium group of Arg 13 and between the cluster sulfide S2 atom and the amide nitrogens of Met 56, Asn 57, Ala 58, and Cys 59 are shown as dotted lines.

In addition to the native structure, the crystal structures of two variants of this protein in which a [2Fe-2S] cysteine ligand was substituted with a serine (Cys55Ser and Cys59Ser) were also determined to 1.25 Å and 1.05 Å, respectively (PDB IDs 1m2b and 1m2d, Figure 3). These high resolution structures provided us with metric details of unprecedented accuracy for serine-ligated iron-sulfur clusters in proteins. (Yeh et al., J. Biol. Chem., 2002)

Figure 3. The [2Fe-2S] cluster, its ligands, and local secondary structure showing the shorter bond formed between (a) Fe2 and the Ser55 Oγ atom in the C55S structure and (b) Fe2 and the Ser59 Oγ atom in the C59S structure. In both figures, simulated-annealing 2|Fo|-|Fc| omit electron density contoured at 1.5 σ level is shown as a cyan mesh.    

Photosynthetic Reaction Center (RC) from the photosynthetic purple bacterium Rhodobacter sphaeroides

The primary process of photosynthesis in photosynthetic bacteria such as Rhodobacter sphaeroides is light-induced trans-membrane charge separation, which occurs in the photosynthetic reaction center (RC), an integral membrane protein-pigment complex (Figure 4a). Upon exposure to light, the RC undergoes a multi-step photocycle, which brings about various conformational changes within the RC structure. To determine whether or not structural change(s) may be associated with one of these steps (the charge-separated D+QA- state), we attempted to induce this state with light, cryogenically trap it, and solve the RC crystal structure in this state. While no major conformational changes were observed, the structure did reveal some disorder and slight conformational changes around the pocket which holds the QA cofactor (Figure 4b).

Figure 4. (a) Overall structure of stigmatellin-bound RC in the dark-adapted state (DQAS), comprising the L (blue), M (yellow), and H (green) subunits, and the two branches of cofactors: bacteriochlorophylls (purple), bacteriopheophytins (cyan), and primary quinone QA (magenta). The tails of the bacteriochlorphyll and bacteriopheophytin cofactors have been omitted from this figure for clarity. The secondary quinone (QB) is replaced by the herbicide stigmatellin (red). The A branch, through which electron transfer occurs, is on the right side in this figure. (b) Superposition of the DQAS (yellow) and D+QA-S (blue) structures showing the similarity of QA (darker shading) and surrounding residues. Hydrogen bonds formed between QA/QA- and the protein environment are depicted by dashed lines.

Photosynthetic Reaction Center (RC) complexed with its electron donor Cytochrome c2 (cyt c2)

During the photocycle, the RC associates with cytochrome c2 (cyt c2), which is a protein that functions as the electron donor to the RC. The crystal structure of the RC complexed with cytochrome c2 was solved (PDB IDs 1l9b and 1l9j) to determine the positioning and interactions between these two proteins. This structure revealed that the binding interface between the RC and cytochrome c2 consists of two domains: (i) a short-range interaction domain (e.g., consisting of nonpolar interactions and hydrogen bonding) which contributes to the strength and specificity of cytochrome c2 binding to the RC and (ii) a long-range, electrostatic interaction domain which may help steer the two unbound proteins towards the right conformation. The structure also revealed a possible electron transfer pathway between the RC and cytochrome c2. (Axelrod et al., J. Mol. Biol., 2002)

Figure 5. (a) The three-dimensional structure of the cyt c2:RC complex from Rb. sphaeroides showing the location of the bound cyt c2 (lavender), the heme prosthetic group (turquoise), the RC L subunit (yellow), the RC M subunit (blue), the RC H-subunit (green), the RC primary donor (red), and non-heme Fe atom (red). The location of the conformational change in the co-crystal at the N-terminal end of the M subunit is indicated by an arrow. (b) The interface region containing the closest contacts between the RC and bound cyt c2. Side chains on cyt c2 (lavender) interacting with RC side chains are shown. Interaction regions on the L subunit of the RC are in yellow and M subunit in blue. The primary donor on the RC is shown in red. The CBC methyl group on the cyt c2 heme (turquoise) is in van der Waals contact with Tyr L162 on the RC. This region contains many short-range non-polar interactions and is believed to be important for intermolecular electron transfer between reduced cyt c2 and the photo-oxidized donor.

Component A of 2-hydroxyglutaryl-CoA dehydratase from the bacterium Acidaminococcus fermentans

Acidaminococcus fermentans is an anaerobic gram-negative bacterium that can, via a fermentation process, use amino acids as its sole energy source  in order to conserve metabolic energy. A common amino acid that undergoes such a fermentation process is glutamate.  A. fermentans degrades glutamate via the hydroxyglutarate pathway, which involves the syn-elimination of water from (R)-2-hydroxyglutaryl-CoA in a key reaction of the pathway.  This anaerobic process is catalyzed by 2-hydroxyglutaryl-CoA dehydratase, an enzyme with two components (A and D) that reversibly associate during reaction cycles. Component A (CompA), a homodimeric protein of 2x27 kDa, contains a single, bridging [4Fe-4S] cluster and uses the hydrolysis of ATP to deliver an electron to the dehydratase component (CompD), where the electron is used catalytically. The structure of the CompA protein, solved by X-ray crystallography to 3 Å resolution (PDB ID 1hux), is a member of the actin fold family, revealing a similar architecture and nucleotide-binding site. The key differences between CompA and other members of the actin fold family were found to be: (i) the presence of a cluster binding segment, the 'cluster helix'; (ii) the [4Fe-4S] cluster; and (iii) the location of the homodimer interface, which involves the bridging cluster. (Locher et al., J. Mol. Biol., 2001)

Bacterioferritin from the nitrogen-oxidizing bacterium Paracoccus denitrificans

Bacterioferritin (BFR), like its eukaryotic ferritin counterpart, functions as an iron-storage protein that consists of a protein shell and an iron core. It consists of 24 subunits which pack to form a highly symmetrical, approximately spherical protein shell surrounding a central cavity in which several thousands of Fe(III) atoms can be stored as hydrous ferric phosphate. From the crystal structure of P. denitrificans BFR, we observed three different types of iron coordination within the protein: (i) heme iron, (ii) dimetal irons at the ferroxidase center, and (iii) polynuclear irons at the core.

Go back to Overview.