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The studies have shown that in both native and reconstituted [4Fe-4S] FNR, the reaction environment and the presence of [Fe.sup.2} and [Fe.sup.3} chelators have influenced the cluster conversion reactions. The sensitivity of FNR cluster conversion kinetics to chelators has disclosed mechanism by which the cell can fine-tune its response to the onset of aerobic conditions.

Research has been conducted on the bacterioferritins. Iron oxidation and hydrolysis chemistry in Escherichia coli bacterioferritin carried out by means of electrode oximetry/pH are discussed.

The fluorescence intensity of Escherichia coli bacterioferritin (BFR), due to the presence of two tryptophan residues (Trp35 and Trp133), is very sensitive to the iron status of the ferroxidase center and is quenched to different extents by [Fe.sup.2+] and [Fe.sup.3+]. The studies have shown that the fluorescence response is used for monitoring the loss of iron from the ferroxidase center by utilizing [Zn.sup.2+], a competitive inhibitor of [Fe.sup.2+] binding and oxidation.

X-ray crystallographic and kinetic methods were used to gain new insight into the mechanism of mineralization in a bacterioferritin (BFR). The inner surface iron site appears to be significant for the transfer of electrons, derived from [Fe.sup.2+] oxidation in the cavity, to the intrasubunit catalytic center, known as the ferroxidase center.

Results point out that catalysis throughout core formation and core nucleation are mediated by ferroxidase center. Data show that roles of the ferroxidase center and the core surface in core formation depict mechanistic aspects of core formation. Data also bring out key differences between mineralization processes in bacterioferritin and mammalian ferritins.

Examines the iron oxidation and hydrolysis chemistry of Escherichia coli bacterioferritin. Detection of three distinct kinetic phases using electrode oximetry/hydrogen ion concentration statistic; Inhibition of iron oxidation by the presence of zinc 2+ ions; Comparison of the inhibition patterns of phase 2 and phase 3.

In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by the fumarate and nitrate reduction transcriptional regulator FNR. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Exposure to O2 results in the conversion of the cluster to a [2Fe-2S]2+ form, leading to dissociation of the protein into transcriptionally inactive monomers. The [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion proceeds in two steps. Step 1 involves the one-electron oxidation of the cluster, resulting in the release of Fe2+ generating a [3Fe-4S]1+ cluster intermediate, and a superoxide ion. In step 2, the cluster intermediate spontaneously rearranges to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and two sulfide ions. Here, we demonstrate that, in both native and reconstituted [4Fe-4S] FNR, the reaction environment and, in particular, the presence of Fe2+ and/or Fe3+ chelators can influence significantly the cluster conversion reaction. We demonstrate that while the rate of step 1 is largely insensitive to chelators, that of step 2 is significantly enhanced by both Fe2+ and Fe3+ chelators. We show that, for reactions in Fe3+-coordinating phosphate buffer, step 2 is enhanced to the extent that step 1 becomes the rate determining step and the [3Fe-4S]1+ intermediate is no longer detectable. Furthermore, Fe3+ released during this step is susceptible to reduction in the presence of Fe2+ chelators. This work, which may have significance for the in vivo FNR cluster conversion reaction in the cell cytoplasm, provides an explanation for apparently contradictory results reported from different laboratories. [ABSTRACT FROM AUTHOR]

Ferritins are iron-storage proteins capable of holding up to 4500 Fe[SUP3+] ions within a single water-soluble protein shell made from 24 polypeptide chains. The Glu128Arg/Glu135Arg mutants of Escherichia coli and Rhodobacter capsulatus bacterioferritins are unable to associate into 24-meric structures, with dimers of polypeptide chains being their stable forms. The aerobic addition to these of up to 8-10 or 14-20 Fe[SUP2+] ions per dimer, respectively, results in the oxidation of the added Fe[SUP2+] to Fe[SUP3+] Gel permeation chromatography and sedimentation equilibrium studies confirm that the Fe[SUP3+] ions are associated with the polypeptide dimer, and the lack of intense EPR signals from magnetically isolated Fe[SUP3+] ions confirms the formation of one or more antiferromagnetically coupled clusters of Fe[SUP3+] ions. The effect of Fe[SUP3+] chelators on iron-loaded subunit dimers is to remove the Fe[SUP3+] from the protein, but to do so slowly, consistent with it not being merely adventitiously associated with protein. These data provide experimental support for the presence of nucleation centers for the mineral cores in bacterioferritins and indicate that these proteins are not simply acting as vessels in which hydrolysis of Fe[SUP3+] occurs independent from the protein surface. From analyses of X-ray structures and amino acid sequence comparisons, possible nucleation sites are identified. [ABSTRACT FROM AUTHOR]

The [Fe 2 S 2 ]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed ( Out ) and buried ( In ) states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the Out and In states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the In state due to a change in its p K a caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes. Our findings illustrate a novel function of protonation resulting from electron transfer.

Iron-sulfur cluster proteins exhibit a range of physicochemical properties that underpin their functional diversity in biology, which includes roles in electron transfer, catalysis, and gene regulation. Transcriptional regulators that utilize iron-sulfur clusters are a growing group that exploit the redox and coordination properties of the clusters to act as sensors of environmental conditions including O2, oxidative and nitrosative stress, and metabolic nutritional status. To understand the mechanism by which a cluster detects such analytes and then generates modulation of DNA-binding affinity, we have undertaken a combined strategy of in vivo and in vitro studies of a range of regulators. In vitro studies of iron-sulfur cluster proteins are particularly challenging because of the inherent reactivity and fragility of the cluster, often necessitating strict anaerobic conditions for all manipulations. Nevertheless, and as discussed in this Account, significant progress has been made over the past decade in studies of O2-sensing by the fumarate and nitrate reduction (FNR) regulator and, more recently, nitric oxide (NO)-sensing by WhiB-like (Wbl) and FNR proteins. Escherichia coli FNR binds a [4Fe-4S] cluster under anaerobic conditions leading to a DNA-binding dimeric form. Exposure to O2 converts the cluster to a [2Fe-2S] form, leading to protein monomerization and hence loss of DNA binding ability. Spectroscopic and kinetic studies have shown that the conversion proceeds via at least two steps and involves a [3Fe-4S](1+) intermediate. The second step involves the release of two bridging sulfide ions from the cluster that, unusually, are not released into solution but rather undergo oxidation to sulfane (S(0)) subsequently forming cysteine persulfides that then coordinate the [2Fe-2S] cluster. Studies of other [4Fe-4S] cluster proteins that undergo oxidative cluster conversion indicate that persulfide formation and coordination may be more common than previously recognized. This remarkable feature suggested that the original [4Fe-4S] cluster can be restored using persulfide as the source of sulfide ion. We have demonstrated that only iron and a source of electrons are required to promote efficient conversion back from the [2Fe-2S] to the [4Fe-4S] form. We propose this as a novel in vivo repair mechanism that does not require the intervention of an iron-sulfur cluster biogenesis pathway. A number of iron-sulfur regulators have evolved to function as sensors of NO. Although it has long been known that the iron-sulfur clusters of many phylogenetically unrelated proteins are vulnerable to attack by NO, our recent studies of Wbl proteins and FNR have provided new insights into the mechanism of cluster nitrosylation, which overturn the commonly accepted view that the product is solely a mononuclear iron dinitrosyl complex (known as a DNIC). The major reaction is a rapid, multiphase process involving stepwise addition of up to eight NO molecules per [4Fe-4S] cluster. The major iron nitrosyl product is EPR silent and has optical characteristics similar to Roussin's red ester, [Fe2(NO)4(RS)2] (RRE), although a species similar to Roussin's black salt, [Fe4(NO)7(S)3](-) (RBS) cannot be ruled out. A major future challenge will be to clarify the nature of these species.