Peter-Leon Hagedoorn

The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.

A conserved iron-binding site, the ferroxidase center, regulates the vital iron storage role of the ubiquitous protein ferritin in iron metabolism. It is commonly thought that two Fe(II) simultaneously bind the ferroxidase center and that the oxidized Fe(III)-O(H)-Fe(III) product spontaneously enters the cavity of ferritin as a unit. In contrast, in some bacterioferritins and in archaeal ferritins a persistent di-iron prosthetic group in this center is believed to mediate catalysis of core formation. Using a combination of binding experiments and isotopically labeled (57)Fe(II), we studied two systems in comparison: the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus (PfFtn) and the eukaryotic human H ferritin (HuHF). The results do not support either of the two paradigmatic models; instead they suggest a unifying mechanism in which the Fe(III)-O-Fe(III) unit resides in the ferroxidase center until it is sequentially displaced by Fe(II).

Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [K(D)] of 17 +/- 7 pM) and molybdate (K(D) of 11 +/- 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low K(D) values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein.

Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys) (3) ligands

2]2 − and its ability to bind to biological macromolecules should not be overlooked, and may artificially trigger/accelerate Cu(II) reduction.

Characterization of the Chlorate Reductase from Pseudomonas chloritidismutans

A chlorate reductase has been purified from the chlorate-reducing strain Pseudomonas chloritidismutans. Comparison with the periplasmic (per)chlorate reductase of strain GR-1 showed that the cytoplasmic chlorate reductase of P. chloritidismutans reduced only chlorate and bromate. Differences were also found in N-terminal sequences, molecular weight, and subunit composition. Metal analysis and electron paramagnetic resonance measurements showed the presence of iron and molybdenum, which are also found in other dissimilatory oxyanion reductases.

Purification and characterization of a chlorite dismutase from Pseudomonas chloritidismutans

The chlorite dismutase (Cld) of Pseudomonas chloritidismutans was purified from the periplasmic fraction in one step by hydroxyapatite chromatography. The enzyme has a molecular mass of 110 kDa and consists of four 31-kDa subunits. Enzyme catalysis followed Michaelis-Menten kinetics, with Vmax and K(m) values of 443 U mg(-1) and 84 microM, respectively. A pyridine-NaOH-dithionite-reduced Cld revealed a Soret peak at 418 nm, indicative for protoheme IX. The spectral data indicate the presence of 1.5 mol protoheme IX mol(-1) tetrameric enzyme while metal analysis revealed 2.2 mol iron mol(-1) tetrameric enzyme. High concentrations of chlorite resulted in the disappearance of the Soret peak, which coincided with loss in activity. Electron paramagnetic resonance analyses showed an axial high-spin ferric iron signal. Cld was inhibited by cyanide, azide, but not by hydroxylamine or 3-amino-1,2,3-triazole. Remarkably, the activity was drastically enhanced by kosmotropic salts, and chaotropic salts decreased the activity, in accordance with the Hofmeister series. Chlorite conversion in the presence of 18O-labeled water did not result in the formation of oxygen with a mass of 34 (16O-18O) or a mass of 36 ((18)O-(18)O), indicating that water is not a substrate in the reaction and that both oxygen atoms originate from chlorite.

Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO42−) and tungstate (WO42−). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteria.

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.

WOR5, a Novel Tungsten-Containing Aldehyde Oxidoreductase from Pyrococcus furiosus with a Broad Substrate Specificity

WOR5 is the fifth and last member of the family of tungsten-containing oxidoreductases purified from the hyperthermophilic archaeon Pyrococcus furiosus. It is a homodimeric protein (subunit, 65 kDa) that contains one [4Fe-4S] cluster and one tungstobispterin cofactor per subunit. It has a broad substrate specificity with a high affinity for several substituted and nonsubstituted aliphatic and aromatic aldehydes with various chain lengths. The highest catalytic efficiency of WOR5 is found for the oxidation of hexanal (V(max) = 15.6 U/mg, K(m) = 0.18 mM at 60 degrees C). Hexanal-incubated enzyme exhibits S = 1/2 electron paramagnetic resonance signals from [4Fe-4S]1+ (g values of 2.08, 1.93, and 1.87) and W5+ (g values of 1.977, 1.906, and 1.855). Cyclic voltammetry of ferredoxin and WOR5 on an activated glassy carbon electrode shows a catalytic wave upon addition of hexanal, suggesting that ferredoxin can be a physiological redox partner. The combination of WOR5, formaldehyde oxidoreductase, and aldehyde oxidoreductase forms an efficient catalyst for the oxidation of a broad range of aldehydes in P. furiosus.

Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli

A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are 64Cu, 67Cu, 187W, 99Mo, 69Zn, 56Mn, 65Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native–native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope 64Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins.

A Conserved Tyrosine in Ferritin Is a Molecular Capacitor

A highly conserved tyrosine residue of unknown function is present in the vicinity of the di‐iron catalytic center of the ubiquitous iron‐storage protein ferritin. The di‐iron center with a gateway FeII/FeIII‐binding site nearby provides the vital iron‐storage mechanism of the protein. It is believed that, in eukaryotic ferritin, this center catalyzes simultaneous oxidation of two FeII ions, whereas in microbial ferritin it catalyzes simultaneous oxidation of three FeII ions. To understand the role of the conserved tyrosine, we studied the intermediates and products that are formed during catalysis of FeII oxidation in the di‐iron catalytic centers of the hyperthermophilic archaeal Pyrococcus furiosus ferritin and of eukaryotic human H ferritin. Based on our spectroscopic studies and modeling, we propose a merger of the models for eukaryotic and bacterial ferritin into a common mechanism of FeII oxidation in which the conserved tyrosine acts as a single‐electron molecular capacitor to facilitate oxidation of FeII.