Interaction of Bacteriophage λ Protein Phosphatase with Mn(II): Evidence for the Formation of a [Mn(II)]₂ Cluster^†

The interaction of bacteriophage λ protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage λ protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases

Evidence for Differential Binding of Isoniazid by <i>Mycobacterium</i> <i>tuberculosis</i> KatG and the Isoniazid-Resistant Mutant KatG(S315T)

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase−peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position ≈12 Å from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a “type I” substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site

Highly Selective Ligand Binding by <i>Methylophilus methylotrophus</i> Cytochrome <i>c</i>′′

Cytochrome c′′ (cyt c′′) from Methylophilus methylotrophus is unusual insofar as the heme has two axial histidine ligands in the oxidized form but one is detached when the protein is reduced. Despite cyt c′′ having an axial site available for binding small ligands, we show here that only NO binds readily to the ferrous cyt c′′. Binding of CO, as well as CN–, on the other hand requires considerable structural reorganization, or reduction of the disulfide bridge close to the heme. Standard free energies for the binding of NO and CO reveal high selectivity of the ferrous cyt c′′ for NO, indicating its putative physiological role. In this work, we characterize in detail the kinetics of NO binding and the structural features of the Fe2+–NO adduct by stopped-flow and resonance Raman spectroscopy, respectively

Redox Properties of Lysine- and Methionine-Coordinated Hemes Ensure Downhill Electron Transfer in NrfH₂A₄ Nitrite Reductase

The multiheme NrfHA nitrite reductase is a menaquinol:nitrite oxidoreductase that catalyzes the 6-electron reduction of nitrite to ammonia in a reaction that involves eight protons. X-ray crystallography of the enzyme from Desulfovibrio vulgaris revealed that the biological unit, NrfH₂A₄, houses 28 <i>c</i>-type heme groups, 22 of them with low spin and 6 with pentacoordinated high spin configuration. The high spin hemes, which are the electron entry and exit points of the complex, carry a highly unusual coordination for <i>c</i>-type hemes, lysine and methionine as proximal ligands in NrfA and NrfH, respectively. Employing redox titrations followed by X-band EPR spectroscopy and surface-enhanced resonance Raman spectroelectrochemistry, we provide the first experimental evidence for the midpoint redox potential of the NrfH menaquinol-interacting methionine-coordinated heme (−270 ± 10 mV, <i>z</i> = 0.96), identified by the use of the inhibitor HQNO, a structural analogue of the physiological electron donor. The redox potential of the catalytic lysine-coordinated high spin heme of NrfA is −50 ± 10 mV, <i>z</i> = 0.9. These values determined for the integral NrfH₂A₄ complex indicate that a driving force for a downhill electron transfer is ensured in this complex

Propofol treatment up-regulates phosphorylated TrkA protein levels in PND14 rats.

<p>Western blot analysis was used to determine the expression of pTrkA in the cortex (A) and thalamus (B). Graphs that show changes in protein levels are accompanied by representative immunoblots. The data are expressed as percentages relative to the respective controls (mean ± SEM): *p<0.05 vs. control.</p

Propofol treatment did not induce neurodegeneration even with detected caspase-3 active fragment in PND14 rats.

<p>Expression of the caspase-3 active fragment in the cortex (A) and thalamus (B). The data are expressed as percentages relative to the respective control (mean ± SEM), *p<0.05 vs. control. (C) Fluoro-Jade B staining (left panel), Hoechst 33258 staining (middle panel) and merged images (right panel) of representative brain sections of the cortex and the thalamus from control-(1^st and 3^rd row, respectively) and propofol-treated (16 h) (2^nd and 4^th row, respectively) PND14 animals. PND7 rats that served as a positive controls were treated with two i.p. doses (0.5 mg/kg) of (+) MK-801 and killed 24 h after the first administration of the drug. Degenerating neurons are marked with arrows and blood vessels with arrowheads. Scale bar = 20 µm.</p

Propofol treatment differentially affects mature BDNF protein levels in PND14 rats.

<p>Western blot analysis was used to determine the expression of mature BDNF in the cortex (A) and thalamus (B). Graphs that show changes in protein levels are accompanied by representative immunoblots. The data are expressed as percentages relative to the respective controls (mean ± SEM): *p<0.05 vs. control.</p

Propofol treatment differentially affects protein levels of total and phosphorylated Akt in PND14 rats.

<p>Western blot analysis was used to determine the expression of Akt and pAkt (Thr308 and Ser473) kinase in the cortex (A) and thalamus (B). Each graph is accompanied by representative immunoblots. The data are expressed as percentages relative to the respective control (mean ± SEM): *p<0.05 vs. control.</p

Propofol treatment differentially affects protein levels of total and phosphorylated ERK1/2 in PND14 rats.

<p>Western blot analysis was used to determine the expression of ERK1/2 and pERK1/2 kinase in the cortex (A) and in the thalamus (B). Each graph is accompanied by representative immunoblots. The data are expressed as percentages relative to the respective control (mean ± SEM): *p<0.05 vs. control.</p

Propofol treatment down-regulates mature NGF protein levels in PND14 rats.

<p>Western blot analysis was used to determine the expression of mature NGF in the cortex (A) and thalamus (B). Graphs that show changes in protein levels are accompanied by representative immunoblots. The data are expressed as percentages relative to the respective controls (mean ± SEM): *p<0.05 vs. control.</p