Search

The reaction of protein-bound iron-sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2 (NO)4 (Cys)2 ]) and Roussin's Black Salt (RBS, [Fe4 (NO)7 S3 ]. In the latter case, the absence of 32 S/34 S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of [O.sub.2], FNR binds a [[4Fe-4S].sup.2+] cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of [O.sub.2], FNR is converted into a monomeric, non-DNA-binding form containing a [[2Fe-2S].sup.2+] cluster. The reaction of the [[4Fe-4S].sup.2+] cluster with [O.sub.2] has been shown to proceed via a 2-step process, an [O.sub.2]-dependent 1-electron oxidation to yield a [[3Fe-4S].sup.+] intermediate with release of I [Fe.sup.2+] ion, followed by spontaneous rearrangement to the [[2Fe-2S].sup.2+] form with release of 1 [Fe.sup.3+] and 2 [S.sup.2-] ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [[4Fe-4S].sup.2+] cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [[4Fe-4S].sup.2+] cluster in the presence of [O.sub.2]. Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with [O.sub.2] is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of [O.sub.2] interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo [O.sub.2] concentrations. gene regulation | iron-sulfur | oxygen sensing | oxidation | transcriptional regulation

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.

The site directed nitroxide spin labeling (SDSL) of cysteine residues were used to search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome b[o.sub.3], Escherichia coli. The kinetic studies of mutants demonstrate that the redox gating of protons occurs deep within the D channel close to the heme-copper site implies that no motion is transmitted to the ends of the helices.

Results show that the water-soluble domain of the nitric oxide reductase subunit C exhibits a alpha-helical structure and contains a partially solvent-exposed heme. The heme iron is coordinated by histidine and methionine ligands in both. Data suggest that the soluble domain of subunit C interacts with the catalytic subunit B.

A magneto-optical study is undertaken of the mixed-valence single-molecule magnet [MnIV (sub 4)MnIII (sub 8) O12 L16] in which the ligands, L, are acetate (Mn12Ac) or the long-chain carboxylic acid, C14 H-29COOH(Mn12C15) that confers better solubility in organic solvents. The optical polarization and the selection rules for field magnetic circular dichroism (MCD) select a subset of molecular orientations with respect to the direction of field.

The crystal structure of the periplasmic penta-heme cytochrome c nitrite reductase of Escherichia coli shows that four hemes of the enzyme exhibit bis-histidine axial heme-iron ligation with the catalytic heme-iron showing a distal lysine ligand and a proximal oxygen ligand. The spectroscopic signals and redox potentials of individual hemes are discussed.

The FnrP is an orthologue of the Escherichia coli transcription factor FNR, which turns on target genes in response to anoxia. Oxygen inactivates the FnrP by disassembling an iron-sulfur cluster whose ligands are cysteines. Data indicate that Cys-14, 17, 25, and 113 are involved in cluster ligation.

Research illustrates the interaction of carbon monoxide with the dinuclear center of reduced bacterrial nitric oxide reductase. Results reveal that the ligand binds to iron(II) heme b(sub)3 forming a stable complex, which suggests differences in the organization of this active site from other heme-copper oxidase superfamily.

The blue copper protein azurin was studied using optically detected electron paramagnetic resonance spectroscopy.

The action of bacterial nitric oxide reductase (NOR) on nitric oxide and nitrous oxide plays an integral role in the completion of the two-electron process undergone by these compounds. NOR is considered a diverged member of the heme-copper oxidase group since it exhibits a distinct electronic absorption spectrum. Experimental data derived from a Pavacocus denitrificans strain suggest that the oxygen reducing activity manifested by heme-copper oxidases is significantly attributed to the substitution of copper for non-heme iron in the dinuclear center.

The spectroscopic data indicate that paracoccus denitrificans periplasmic nitrate reductase exhibit distinct molybdenum (Mo) oxidation states comprised of Mo(VI), (V), and (IV) and possesses a Mo-cysteine ligand. A model is presented for the enzyme catalysis and Mo coordination during enzyme turnover.

A study was conducted on the fully oxidized nitric oxide reductase (NOR) from Pseudomonas stutzeri. The findings indicate a spin-spin interaction between the heme and another paramagnet as well as an unusual near-infrared MCD. Moreover, the results suggest that NOR is structurally related to heme-copper oxidases and that it has a heme/non-heme iron spin-coupled pair at the active site. Thus, the nature of the metal ion paired with the heme in the active site may be essential in determining the specificity of these enzymes.

Halide metathesis with silver nitrite can be used for either stepwise or complete substitution of all three chloride ligands in the iron(III) compound (NEt4)(HB(3,5-Me2pz)3FeCl) to produce the complexes (NEt4)(HB(3,5-Me2pz)3Fe(ONO)xCl3-x), x = 1 - 3, and (NEt4)(HB(3,5-Me2pz)3Fe(eta2-ONO)Cl). The above compounds were characterized by elemental analyses, electrochemistry and spectroscopy. The crystal structures of the ferric complexes had monodentate, O-bound nitrito ligands.

The properties of the cytochromes cdsub1 from Pseudomonas stutzeri and Thiosphaera pantotropha have been compared, using MCD and EPR techniques. The results are consistent with previous indications that enzymes from different sources do not have identical heme iron ligands in the oxidized state, indicating there there are several classes of cytochrome cdsub1.

Electronic absorption, MCD and EPR spectroscopy have been used to study the reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli. Two distinct species were formed and it was revealed that broad EPR signals are lost. Heme o stays in the high-spin FeIII state during titration, and the results are consistent with binding of two NO molecules at CuBII.

The reactive 3Fe cluster in the 7Fe ferredoxin from Desulfovibrio africanus, incorporating Fe and other metals to to form cubanes (M3Fe-4S) has a non-cystein ligation to the added metal (M). Connectivities among interconversion with different M, the influence of pH and the reactivities of certain cluster oxidation levels have been studied using electrochemical and spectroscopic methods.