Chronic inflammatory diseases place a heavy social and economic burden on the resources of many nations, but the number of safe and effective treatments is limited. To date, the major research effort has concentrated on those mediators responsible for the initiation and maintenance of the pathological process. In contrast, little attention has been focused on endogenous factors responsible for the resolution of the inflammation. Heme oxygenase ((HO); EC 1.14.99.3) is the rate-limiting enzyme in the catabolism of heme to biliverdin (which is converted to bilirubin by biliverdin reductase), free iron and carbon monoxide (CO). Two isoforms of HO have been characterized, the constitutive isoform, HO-2, which is the major isoform present under physiological conditions, and the stress-induced isoform, HO-1, which has also been classified as heat-shock protein 32K (ref. 1). Increases in HO activity have been implicated in tissue protection against oxidative stress. In this communication, we describe the effects of modulating HO during an acute complement-dependent inflammatory response. Elevation of this enzyme resulted in a striking suppression, whereas inhibition of the enzyme led to a potentiation of the inflammatory response. Such novel enzyme modulation has application on the one hand to the treatment of inflammatory diseases and on the other hand to immnosuppressed states in which the impaired ability to mount an adequate inflammatory response may result in death from opportunistic infections.

We have determined the solution structure of a 15-mer boxB RNA hairpin complexed with a 20-mer basic peptide of the N protein involved in bacteriophage P22 transcriptional antitermination. Complex formation involves adaptive binding with the N peptide adopting a bent alpha-helical conformation that packs tightly through hydrophobic and electrostatic interactions against the major groove face of the boxB RNA hairpin, orienting the open opposite face for potential interactions with host factors and/or RNA polymerase. Four nucleotides in the boxB RNA hairpin pentaloop form a stable GNRA like tetraloop structural scaffold on complex formation, allowing the looped out fifth nucleotide to make extensive hydrophobic contacts with the bound peptide. The guanidinium group of a key arginine is hydrogen-bonded to the guanine in a loop-closing sheared G.A mismatch and to adjacent backbone phosphates. The identified intermolecular contacts account for the consequences of N peptide and boxB RNA mutations on bacteriophage transcriptional antitermination.

Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S0 is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.

IscU from Escherichia coli, the scaffold protein for iron-sulfur cluster biosynthesis and delivery, populates a complex energy landscape. IscU exists as two slowly interconverting species: one (S) is largely structured with all four peptidyl–prolyl bonds trans; the other (D) is partly disordered but contains an ordered domain that stabilizes two cis peptidyl–prolyl peptide bonds. At pH 8.0, the S-state is maximally populated at 25 °C, but its population decreases at higher or lower temperatures or at lower pH. The D-state binds preferentially to the cysteine desulfurase (IscS), which generates and transfers sulfur to IscU cysteine residues to form persulfides. The S-state is stabilized by Fe–S cluster binding and interacts preferentially with the DnaJ-type co-chaperone (HscB), which targets the holo-IscU:HscB complex to the DnaK-type chaperone (HscA) in its ATP-bound from. HscA is involved in delivery of Fe–S clusters to acceptor proteins by a mechanism dependent on ATP hydrolysis. Upon conversion of ATP to ADP, HscA binds the D-state of IscU ensuring release of the cluster and HscB. These findings have led to a more complete model for cluster biosynthesis and delivery.