Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells.

The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite.

Mass instability in isolated recombinant FixL heme domains of Bradyrhizobium japonicum.

Several recombinant Bradyrhizobium japonicum FixL heme domains (BjFixLH) have been characterized and their temporal mass stabilities assessed by MALDI-TOF mass spectrometry. The intact heme domains all bound heme and gave normal UV-visible spectra, indicating that they were correctly assembled. Proteins produced at Washington State University included a parent 131-amino acid "full-length heme domain" (FLHD) of primary sequence T140-Q270 (BjFixLH140-270), a histidine-tagged analogue containing an N-terminal extension, and five different terminus-truncated variants.

Pathways for intracellular generation of oxidants and tyrosine nitration by a macrophage cell line.

Two transformed murine macrophage cell lines (RAW 264.7 ATCC TIB-71 and CRL-2278) were examined for oxidant production at various times following activation by using a set of fluorescence and ESR-active probes. Stimulation with a soluble agonist or activation with bacterial lipopolysaccharide plus gamma-interferon caused only very small initial increases in O2 consumption above basal rates; however, at 2-4 h post-activation, respiration increased to 2-3-fold and remained at these elevated levels over the subsequent lifetime of the cell (20-30 h).

Characterization of conformational changes coupled to ligand photodissociation from the heme binding domain of FixL.

Using transient absorption spectroscopy and photoacoustic calorimetry (PAC), we have characterized carbon monoxide photodissociation and rebinding to two forms of the heme domain of Bradyrhizobium japonicum FixL. Transient absorption results for the complete heme domain (FixL residues 140-270) and a truncated heme domain (missing 11 residues on the N-teminal end and 14 amino acid residues on the C-terminal end of the full length heme domain) show similar rates for ligand rebinding to the five-coordinate heme domain and the absence of any transient intermediate on a microsecond time scale. Results from PAC studies show that both the truncated and complete heme domains undergo a contraction upon ligand photolysis.

Recombinant PAS-heme domains of oxygen sensing proteins: high level production and physical characterization.

Details of a high-level recombinant production method for the heme-PAS domains of heme oxygen sensing proteins from Sinorhizobium meliloti (Sm) (formerly Rhizobium meliloti, Rm), Bradyrhizobium japonicum (Bj), and Escherichia coli (Ec) are described. Using a newly proposed, concise, and unambiguous naming system (also described here) these proteins are: SmFixLH(128-264), BjFixLH(140-270), and EcDosH(1-147). In addition, high-level production of BjFixL(140-505), the soluble full-length protein containing both heme (oxygen sensing) and kinase (catalytic) domains is described.

Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils.

Escherichia coli were transformed by electroporation to introduce a plasmid harboring a GFP gene-containing vector. The fluorescence of the purified GFP isolated from the transformant was quenched by myeloperoxidase (MPO)-generated HOCl, by peroxynitrous acid (ONOOH) and by enzymatically or radiolytically generated NO(2)(.) but not by other putative neutrophil-generated oxidants.

Insights into signal transduction involving PAS domain oxygen-sensing heme proteins from the X-ray crystal structure of Escherichia coli Dos heme domain (Ec DosH).

The X-ray crystal structure of the Escherichia coli (Ec) direct oxygen sensor heme domain (Ec DosH) has been solved to 1.8 A using Fe multiple-wavelength anomalous dispersion (MAD), and the positions of Met95 have been confirmed by selenomethionine ((Se)Met) MAD. Ec DosH is the sensing part of a larger two-domain sensing/signaling protein, in which the signaling domain has phosphodiesterase activity.

Cloning, purification, crystallization and preliminary X-ray analysis of DOS heme domain, a new heme oxygen sensor in Escherichia coli.

The heme-containing PAS domain of the direct oxygen-sensor protein (DOS(H)), a bona fide oxygen-sensor protein, has been cloned from Escherichia coli strain K12 and successfully purified. The oxidized form of this protein was crystallized by the hanging-drop method with a PEG 8000-based precipitant. Preliminary X-ray diffraction studies of the PAS-domain crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.