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. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking.

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. The smallest of these was a 106-amino acid "core PAS heme domain" with primary sequence T151-L256. All variants except for the smallest exhibited significant mass instability, assessed by MALDI-TOF mass spectrometry, that was apparent within 1-16 days standing in a sterile environment at room temperature. Two full-length heme domains expressed independently in geographically remote laboratories (Northern Illinois University and JILA, University of Colorado) also exhibited this mass instability. A mass loss of as much as approximately 25% of the starting mass has been observed, which could explain the "missing" terminal amino acids in published crystal structures. This work documents the phenomenon and its persistence despite (i) sample sterilization, (ii) protease inhibitors, (iii) primary sequence variations, (iv) the presence or absence of ferriheme ligands, and (v) the presence or absence of O2.

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). Oxidation reactions were confined primarily within the cell, as was demonstrated by using phagocytosable dichlorodihydrofluorescein-conjugated latex beads and cyclic hydroxylamines with differing membrane permeabilities. From the intrinsic reactivities of these probes and the time course of their oxidations, one infers the induction of apparent peroxidase activity beginning at approximately 2 h post-activation coinciding with the increase in overall respiratory rate; this acquired capability was accompanied by accumulation of a stable horseradish peroxidase-reactive oxidant, presumably H2O2, in the extracellular medium. Nitrite ion rapidly accumulated in the extracellular medium over a period of 5-8 h post-activation in both cell lines, indicating the presence of active nitric oxide synthase (iNOS) during that period. Prostaglandin endoperoxide H synthase (COX-2) activity was detected at 15-20 h post-activation by the use of a sensitive peroxide assay in conjunction with a COX-2 specific inhibitor (DuP-697). Superoxide formation was detected by reaction with hydroethidine within the first hour following activation, but not thereafter. Consistent with the absence of significant respiratory stimulation, the amount of O2*- formed was very small; comparative reactions of cyclic hydroxylamine probes indicated that virtually none of the O2*- was discharged into the external medium. Myeloperoxidase (MPO) activity was probed at various times post-activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCl in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from approximately 6.5 to approximately 8.2 over a 20 h post-phagocytosis period. The cumulative data suggest that activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O2*- playing a minimal role in the direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO2- is used by COX-2 to produce NO2* as a terminal microbicidal oxidant and nitrating agent.

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. In addition, CO photolysis from the complete heme domain gives rise to an intermediate with a lifetime of approximately 150 ns which is absent in the truncated heme domain. We attribute the 150 ns phase to ligand release to the solvent which may be accelerated in the case of the truncated domain. The initial contraction is attributed to changes in the charge distribution due to reorganization of the surface salt bridge formed between Glu182 and Arg227 or possibly to reorientation of Arg206. Changes in the charge distribution may play an important role in communication between the sensor domain and the regulatory domain and thus may be part of the signal transduction pathway.

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. Using an IPTG-inducible pET/BL21 expression system and a rapid, two-column purification has resulted in increased yields of 3- to 17-fold over literature values. The recombinant proteins are highly pure as judged by SDS-PAGE, MALDI-TOF mass spectrometry, and a UV-visible purity index. To our knowledge, this work includes the first mass spectrometry analysis of any PAS-heme protein and provides high-resolution confirmation of each protein's identity. These production and characterization improvements make possible future spectroscopic and dynamics studies designed to elucidate the intramolecular/interdomain signal that follows heme-domain oxygen dissociation.

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. Fluorescence from the bacterium was effectively quenched by HOCl but not peroxynitrite, oxidizing radicals derived from its O-O bond homolysis, or the other oxidants under study. Exposure of serum-opsonized bacteria to human neutrophils resulted in extensive loss of GFP fluorescence; fluorescence microscopy revealed that phagocytosed bacteria were completely quenched but that bacteria remaining in the external media were unquenched. Addition of sodium azide to the medium to inhibit MPO prevented neutrophil-mediated fluorescence quenching. Because the amount of HOCl required to inhibit bacterial fluorescence was an order of magnitude greater than required to inhibit colonial growth, these results imply that sufficient HOCl was formed within the neutrophil phagosome to kill the microbe.

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. The asymmetric unit of the crystal lattice contains a dimer comprised of two differently ligated heme domain monomers. Except for the heme ligands, the monomer heme domains are identical. In one monomer, the heme is ligated by molecular oxygen (O(2)), while in the other monomer, an endogenous Met95 with S --> Fe ligation replaces the exogenous O(2) ligand. In both heme domains, the proximal ligand is His77. Analysis of these structures reveals sizable ligand-dependent conformational changes in the protein chain localized in the FG turn, the G(beta)-strand, and the HI turn. These changes provide insight to the mechanism of signal propagation within the heme domain following initiation due to O(2) dissociation.

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 = 46.1, b = 68.1, c = 82.6 A. A complete diffraction data set was collected to 1.9 A for MAD phasing. The electron-density map shows two molecules in an asymmetric unit and a unique six-coordination of the heme iron.