Allison Lewin

Iron detoxification properties of Escherichia coli bacterioferritin. Attenuation of oxyradical chemistry.

Bacterioferritin (EcBFR) ofEscherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the “ferroxidase center.” The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H2O2 as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H2O2 at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H2O2 corresponds to [Fe(II)2-P]Z + H2O2→ [Fe(III)2O-P]Z + H2O, where [Fe(II)2-P]Z represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)2O-P]Z a μ-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe2+ + H2O2 + 2H2O → 2FeOOH(core) + 4H+, where two Fe(II) are again oxidized by one H2O2. Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O2 is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H2O2produced from O2 is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H2O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H2O2, consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H2O2, thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.

Structural Basis for Iron Mineralization by Bacterioferritin

Ferritin proteins function to detoxify, solubilize and store cellular iron by directing the synthesis of a ferric oxyhydroxide mineral solubilized within the proteins central cavity. Here, through the application of X-ray crystallographic and kinetic methods, we report significant new insight into the mechanism of mineralization in a bacterioferritin (BFR). The structures of nonheme iron-free and di-Fe(2+) forms of BFR showed that the intrasubunit catalytic center, known as the ferroxidase center, is preformed, ready to accept Fe(2+) ions with little or no reorganization. Oxidation of the di-Fe(2+) center resulted in a di-Fe(3+) center, with bridging electron density consistent with a mu-oxo or hydro bridged species. The mu-oxo bridged di-Fe(3+) center appears to be stable, and there is no evidence that Fe(3+)species are transferred into the core from the ferroxidase center. Most significantly, the data also revealed a novel Fe(2+) binding site on the inner surface of the protein, lying approximately 10 A directly below the ferroxidase center, coordinated by only two residues, His46 and Asp50. Kinetic studies of variants containing substitutions of these residues showed that the site is functionally important. In combination, the data support a model in which the ferroxidase center functions as a true catalytic cofactor, rather than as a pore for the transfer of iron into the central cavity, as found for eukaryotic ferritins. The inner surface iron site appears to be important for the transfer of electrons, derived from Fe(2+) oxidation in the cavity, to the ferroxidase center. Bacterioferritin may represent an evolutionary link between ferritins and class II di-iron proteins not involved in iron metabolism.

Formation of protein-coated iron minerals

The ability of iron to cycle between Fe(2+) and Fe(3+) forms has led to the evolution, in different forms, of several iron-containing protein cofactors that are essential for a wide variety of cellular processes, to the extent that virtually all cells require iron for survival and prosperity. The redox properties of iron, however, also mean that this metal is potentially highly toxic and this, coupled with the extreme insolubility of Fe(3+), presents the cell with the significant problem of how to maintain this essential metal in a safe and bioavailable form. This has been overcome through the evolution of proteins capable of reversibly storing iron in the form of a Fe(3+) mineral. For several decades the ferritins have been synonymous with the function of iron storage. Within this family are subfamilies of mammalian, plant and bacterial ferritins which are all composed of 24 subunits assembled to form an essentially spherical protein with a central cavity in which the mineral is laid down. In the past few years it has become clear that other proteins, belonging to the family of DNA-binding proteins from starved cells (the Dps family), which are oligomers of 12 subunits, and to the frataxin family, which may contain up to 48 subunits, are also able to lay down a Fe(3+) mineral core. Here we present an overview of the formation of protein-coated iron minerals, with particular emphasis on the structures of the protein coats and the mechanisms by which they promote core formation. We show on the one hand that significant mechanistic similarities exist between structurally dissimilar proteins, while on the other that relatively small structural differences between otherwise similar proteins result in quite dramatic mechanistic differences.

Iron detoxification properties of E.coli Bacterioferritin: Attenuation of oxyradical chemistry

Bacterioferritin (EcBFR) ofEscherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the “ferroxidase center.” The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H2O2 as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments. Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H2O2 at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry. The oxidation reaction with H2O2 corresponds to [Fe(II)2-P]Z + H2O2→ [Fe(III)2O-P]Z + H2O, where [Fe(II)2-P]Z represents a diferrous ferroxidase center complex of the protein P with net charge Z and [Fe(III)2O-P]Z a μ-oxo-bridged diferric ferroxidase complex. The mineralization reaction is given by 2Fe2+ + H2O2 + 2H2O → 2FeOOH(core) + 4H+, where two Fe(II) are again oxidized by one H2O2. Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O2 is used as the oxidant in both the ferroxidation and mineralization reactions. Most of the H2O2produced from O2 is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H2O. EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H2O2, consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H2O2, thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry.

PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity

PrrC from Rhodobacter sphaeroides provides the signal input to a two‐component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry 39, 2052‐2062; Oh, J.‐I. and Kaplan, S. (2000) EMBO J. 19, 4237‐4247). It is also a homologue of eukaryotic Sco proteins and each has a C‐x‐x‐x‐C‐P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis of the CuA centre of respiratory cytochrome oxidase. Overexpression and purification of a water‐soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal‐free rPrrC (apo‐PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV‐visible spectrum, having absorbance with a λ max of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal‐free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O2 sensitivity are involved in copper binding in reduced PrrC since the same copper‐loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol‐disulfide oxidoreductase function and a copper‐binding role.

The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion

The ferric uptake regulator (Fur) of Pseudomonas aeruginosa was expressed in Escherichia coli in its native form and as a fusion to the maltose-binding protein (MBP). Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding. The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays. As-prepared native Fur contained small amounts of Zn(2+) that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn(2+) ion per monomer. Thus, the P. aeruginosa Fur can probably accommodate a single Zn(2+) ion bound to the metal-sensing site. The single cysteine residue of P. aeruginosa Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the E. coli protein, and is believed to provide one of the ligands to a structural Zn(2+) ion. This cysteine residue was shown to be dispensable for the in vivo activity of P. aeruginosa Fur, which is consistent with the suggestion that the P. aeruginosa protein does not contain a structural Zn(2+) ion. Members of the Fur family contain a highly conserved His-His-Asp-His motif. Alanine substitutions of residues in this motif showed His-87 and His-89 of P. aeruginosa Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable.

Molecular basis for specificity of the extracytoplasmic thioredoxin ResA

ResA, an extracytoplasmic thioredoxin from Bacillus subtilis, acts in cytochrome c maturation by reducing the disulfide bond present in apocytochromes prior to covalent attachment of heme. This reaction is (and has to be) specific, as broad substrate specificity would result in unproductive shortcircuiting with the general oxidizing thioredoxin(s) present in the same compartment. Using mutational analysis and subsequent biochemical and structural characterization of active site variants, we show that reduced ResA displays unusually low reactivity at neutral pH, consistent with the observed high pKa values >8 for both active site cysteines. Residue Glu80 is shown to play a key role in controlling the acid-base properties of the active site. A model in which substrate binding dramatically enhances the reactivity of the active site cysteines is proposed to account for the specificity of the protein. Such a substratemediated activation mechanism is likely to have wide relevance for extracytoplasmic thioredoxins.

Crystal Structure and Biophysical Properties of Bacillus subtilis BdbD AN OXIDIZING THIOL:DISULFIDE OXIDOREDUCTASE CONTAINING A NOVEL METAL SITE

BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca2+, at an inter-domain position some 14 Å away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as −75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pKa, consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca2+-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca2+-depleted BdbD was significantly less stable than reduced Ca2+-containing protein, and the midpoint reduction potential was shifted by approximately −20 mV, suggesting that Ca2+ functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.

Effects of substitutions in the CXXC active-site motif of the extracytoplasmic thioredoxin ResA

The thiol-disulfide oxidoreductase ResA from Bacillus subtilis fulfils a reductive role in cytochrome c maturation. The pK(a) values for the CEPC (one-letter code) active-site cysteine residues of ResA are unusual for thioredoxin-like proteins in that they are both high (>8) and within 0.5 unit of each other. To determine the contribution of the inter-cysteine dipeptide of ResA to its redox and acid-base properties, three variants (CPPC, CEHC and CPHC) were generated representing a stepwise conversion into the active-site sequence of the high-potential DsbA protein from Escherichia coli. The substitutions resulted in large decreases in the pK(a) values of both the active-site cysteine residues: in CPHC (DsbA-type) ResA, DeltapK(a) values of -2.5 were measured for both cysteine residues. Increases in midpoint reduction potentials were also observed, although these were comparatively small: CPHC (DsbA-type) ResA exhibited an increase of +40 mV compared with the wild-type protein. Unfolding studies revealed that, despite the observed differences in the properties of the reduced proteins, changes in stability were largely confined to the oxidized state. High-resolution structures of two of the variants (CEHC and CPHC ResA) in their reduced states were determined and are discussed in terms of the observed changes in properties. Finally, the in vivo functional properties of CEHC ResA are shown to be significantly affected compared with those of the wild-type protein.

Monitoring the iron status of the ferroxidase center of Escherichia coli bacterioferritin using fluorescence spectroscopy.

Ferritins solubilize and detoxify the essential metal iron through formation of a ferric mineral within the proteins central cavity. Key to this activity is an intrasubunit catalytic dinuclear iron center called the ferroxidase center. Here we show that the fluorescence intensity of Escherichia coli bacterioferritin (BFR), due to the presence of two tryptophan residues (Trp35 and Trp133) in each of the 24 subunits, is highly sensitive to the iron status of the ferroxidase center and is quenched to different extents by Fe2+ and Fe3+. Recovery of the quench following oxidation of Fe2+ to Fe3+ at the ferroxidase center was not observed, indicating that the di-Fe3+ form of the center is stable. Studies of the single-tryptophan variants W35F and W133F showed that Trp133, which lies approximately 10 A from the ferroxidase center, is primarily responsible for the observed fluorescence sensitivity to iron, while studies of a stable E. coli BFR subunit dimer demonstrated that the observed quench properties are principally derived from the interaction of iron with tryptophan residues within the subunit dimer. A double-tryptophan variant (W35F/W133F) was found to exhibit fluorescence from the seven tyrosine residues present in each subunit, which was also sensitive to the iron status of the ferroxidase center. Finally, we demonstrate using Zn2+, a potent competitive inhibitor of Fe2+ binding and oxidation, that the fluorescence response can be used to monitor the loss of iron from the ferroxidase center.