Probing Ligand Exchange in the P450 Enzyme CYP121 from <i>Mycobacterium tuberculosis</i>: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature

CYP121 is a cytochrome P450 enzyme from <i>Mycobacterium tuberculosis</i> that catalyzes the formation of a C–C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme–substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O₂ binding. Here we employed sodium cyanide to probe the metal–ligand exchange of the enzyme and the enzyme–substrate complex. The cyano adducts were characterized by UV–vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme–substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121­(cYY)­CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121­(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (<i>k</i>_on (CN)) but a much more stable cyanide adduct with 3 orders of magnitude slower <i>k</i>_off (CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme–substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism

Iron Pincer Complexes Incorporating Bipyridine: A Strategy for Stabilization of Reactive Species

Treatment of FeCl₂(thf)_1.5 with the pincer ligand bis­(dicyclohexylphosphinomethyl)­pyrrolide (^CyPNP) in the presence of 2,2′-bipyridine (bipy) affords the six-coordinate complex [FeCl­(bipy)­(^CyPNP)]. The chloride complex exhibits spin-crossover behavior and serves as a starting point for a series of bipy-coordinated iron­(II) pincer compounds, including the hydride species [FeH­(bipy)­(^CyPNP)]. Chemical reduction of [FeCl­(bipy)­(^CyPNP)] generates the new complex [Fe­(bipy)­(^CyPNP)], which is found to be consistent with a structure featuring iron­(I) as opposed to a bipy radical anion. Reactivity studies with the bipy complexes demonstrate that they are more prone to migratory insertion and β-hydrogen elimination reactions than the corresponding dicarbonyl analogues and that in certain instances the bipy ligand can be sequestered by treatment with a Lewis acid. These observations demonstrate that coordination of bipyridine to pincer-ligated iron­(II) species provides a means of stabilizing otherwise unstable compounds while permitting a higher degree of reactivity than is possible with carbonyl coligands

High-Frequency/High-Field Electron Paramagnetic Resonance and Theoretical Studies of Tryptophan-Based Radicals

Tryptophan-based free radicals have been implicated in a myriad of catalytic and electron transfer reactions in biology. However, very few of them have been trapped so that biophysical characterizations can be performed in a high-precision context. In this work, tryptophan derivative-based radicals were studied by high-frequency/high-field electron paramagnetic resonance (HFEPR) and quantum chemical calculations. Radicals were generated at liquid nitrogen temperature with a photocatalyst, sacrificial oxidant, and violet laser. The precise <i>g</i>-anisotropies of l- and d-tryptophan, 5-hydroxytryptophan, 5-methoxytryptophan, 5-fluorotryptophan, and 7-hydroxytryptophan were measured directly by HFEPR. Quantum chemical calculations were conducted to predict both neutral and cationic radical spectra for comparison with the experimental data. The results indicate that under the experimental conditions, all radicals formed were cationic. Spin densities of the radicals were also calculated. The various line patterns and <i>g</i>-anisotropies observed by HFEPR can be understood in terms of spin-density populations and the positioning of oxygen atom substitution on the tryptophan ring. The results are considered in the light of the tryptophan and 7-hydroxytryptophan diradical found in the biosynthesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase

Backbone Dehydrogenation in Pyrrole-Based Pincer Ligands

Treatment of both [CoCl­(^<i>t</i>BuPNP)] and [NiCl­(^<i>t</i>BuPNP)] (^<i>t</i>BuPNP = anion of 2,5-bis­((di-<i>tert</i>-butylphosphino)­methyl)­pyrrole) with one equivalent of benzoquinone affords the corresponding chloride complexes containing a dehydrogenated PNP ligand, ^<i>t</i>BudPNP (^<i>t</i>BudPNP = anion of 2,5-bis­((di-<i>tert</i>-butylphosphino)­methylene)-2,5-dihydropyrrole). Dehydrogenation of PNP to dPNP results in minimal change to steric profile of the ligand but has important consequences for the resulting redox potentials of the metal complexes, resulting in the ability to isolate both [CoH­(^<i>t</i>BudPNP)] and [CoEt­(^<i>t</i>BudPNP)], which are more challenging (hydride) or not possible (ethyl) to prepare with the parent PNP ligand. Electrochemical measurements with both the Co and Ni dPNP species demonstrate a substantial shift in redox potentials for both the M­(II/III) and M­(II/I) couples. In the case of the former, oxidation to trivalent Co was found to be reversible, and subsequent reaction with AgSbF₆ afforded a rare example of a square-planar Co­(III) species. Corresponding reduction of [CoCl­(^<i>t</i>BudPNP)] with KC₈ produced the diamagnetic Co­(I) species, [Co­(N₂)­(^<i>t</i>BudPNP)]. Further reduction of the Co­(I) complex was found to generate a pincer-based π-radical anion that demonstrated well-resolved EPR features to the four hydrogen atoms and lone nitrogen atom of the ligand with minor contributions from cobalt and coordinated N₂. Changes in the electronic character of the PNP ligand upon dehydrogenation are proposed to result from loss of aromaticity in the pyrrole ligand, resulting in a more reducing central amido donor. DFT calculations on the Co­(II) complexes were performed to shed further insight into the electronic structure of the pincer complexes

Additional file 1: of Safety of pazopanib and sunitinib in treatment-naive patients with metastatic renal cell carcinoma: Asian versus non-Asian subgroup analysis of the COMPARZ trial

Conduct of NCT00720941 and NCT01147822 and timing of key study events. Protocol amendment 4 authorized inclusion of patients from NCT01147822 for safety and efficacy analyses. FPFV = first patient first visit. Data from Motzer RJ et al. [8]. Conduct of trials NCT00720941 and NCT01147822. (PDF 111 kb

Elevated <i>Mirc1/Mir17-92</i> cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages

<p>Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the <i>CF transmembrane conductance regulator (CFTR)</i> gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (<i>Mirs</i>), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of <i>Mirs</i> in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the <i>Mirc1/Mir17-92</i> cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between <i>Mirc1/Mir17-92</i> and autophagy gene expression. An <i>in silico</i> study for targets of <i>Mirs</i> that comprise the cluster suggested that the majority of the <i>Mirs</i> target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated <i>Mirs</i>, via restoration of autophagy. In vivo, downregulation of <i>Mir17</i> and <i>Mir20a</i> partially restored autophagy expression and hence improved the clearance of <i>Burkholderia cenocepacia</i>. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.</p