EPR studies of bacterioferritin (BFR), an iron‐storage protein of Escherichia coli [1993, Biochem. J. 292, 47‐56.], have revealed the presence of non‐haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release. When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found. Under certain conditions, an iron dimer was also observed. The reaction of phosphate with NHI species has been investigated. Results point to a function for this anion in core nucleation.
The rsmA gene of Streptomyces coelicolor lies directly upstream of the gene encoding the group 3 sigma factor sigma(M). The RsmA protein is a putative member of the HATPase_c family of anti-sigma factors but is unusual in that it contains seven cysteine residues. Bacterial two-hybrid studies demonstrate that it interacts specifically with sigma(M), and in vitro studies of the purified proteins by native PAGE and transcription assays confirmed that they form a complex. Characterization of RsmA revealed that it binds ATP and that, as isolated, it contains significant quantities of iron and inorganic sulfide, in equal proportion, with spectroscopic properties characteristic of a [2Fe-2S] cluster-containing protein. Importantly, the interaction between RsmA and sigma(M) is dependent on the presence of the iron-sulfur cluster. We propose a model in which RsmA regulates the activity of sigma(M). Loss of the cluster, in response to an as yet unidentified signal, activates sigma(M) by abolishing its interaction with the anti-sigma factor. This represents a major extension of the functional diversity of iron-sulfur cluster proteins.
CyaY, the frataxin homolog of Escherichia coli , is known to regulate ISC iron–sulfur cluster assembly through binding to IscS. It also interacts with the SUF system, through binding to, and attenuating activity of, SufS.
Abstract NsrR from Streptomyces coelicolor ( Sc) regulates the expression of three genes through the progressive degradation of its [4Fe–4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe–4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.
CopA from Bacillus subtilis is a Cu(I)-transporting P-type ATPase involved in resistance to high levels of environmental copper. At its N-terminus are two soluble domains, a and b, that, when generated in isolation from the membrane part, have previously been shown to exhibit unusual Cu(I)-binding behaviour: at >1 Cu(I) per CopAab the protein dimerises, resulting in the formation of a species with luminescence properties characteristic of a solvent-shielded Cu(I) cluster. Further insight into the Cu(I)-binding properties of CopAab are now reported. We demonstrate that the initial binding of Cu(I) occurs with very high affinity (K = -4 x 10(17) M(-1)) and that CopAab can accommodate up to 4 Cu(I) per protein and remains dimeric at higher Cu(I)-loadings. Fitting of UV-visible, near UV CD, fluorescence and luminescence spectroscopic titration data supports a model in which Cu(I) binds sequentially to CopAab, and also provides estimates of the association constants for Cu(I)-binding and dimerisation steps. Finally, low molecular weight thiols are shown not to affect the initial binding of Cu(I), but significantly influence binding at levels >1 Cu(I) per CopAab such that dimerisation is inhibited, though not abolished.
The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake. The bacterioferritin (BFR) of Escherichia coli is a heme-containing iron storage molecule. It is composed of 24 identical subunits, which form a roughly spherical protein shell surrounding a central iron storage cavity. Each of the 12 heme moieties of BFR possesses bis-methionine axial ligation, a heme coordination scheme so far only found in bacterioferritins. Members of the BFR family contain three partially conserved methionine residues (excluding the initiating methionine) and in this study each was substituted by leucine and/or histidine. The Met52 variants were devoid of heme, whereas the Met31 and Met86 variants possessed full heme complements and were spectroscopically indistinguishable from wild-type BFR. The heme-free Met52 variants appeared to be correctly assembled and were capable of accumulating iron both in vivo and in vitro. No major differences were observed in the overall rate of iron accumulation for BFR-M52H, BFR-M52L, and the wild-type protein. The iron contents of the Met52 variants, as isolated, were at least 4 times greater than for wild-type BFR. This study is consistent with the reported location of the BFR heme site at the 2-fold axis and shows that heme is unnecessary for BFR assembly and iron uptake.
Ferritin proteins function to detoxify, solubilize and store cellular iron by directing the synthesis of a ferric oxyhydroxide mineral solubilized within the protein's 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-Fe2+ forms of BFR showed that the intrasubunit catalytic center, known as the ferroxidase center, is preformed, ready to accept Fe2+ ions with little or no reorganization. Oxidation of the di-Fe2+ center resulted in a di-Fe3+ center, with bridging electron density consistent with a μ-oxo or hydro bridged species. The μ-oxo bridged di-Fe3+ center appears to be stable, and there is no evidence that Fe3+species are transferred into the core from the ferroxidase center. Most significantly, the data also revealed a novel Fe2+ binding site on the inner surface of the protein, lying ∼10 Å 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 Fe2+ 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.
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