Journal of Biological Inorganic Chemistry最新文献

Lipid nanoparticles formed with copolymers are a new and increasingly powerful tool for studying membrane proteins, but the extent to which these systems affect the physical properties of the membrane is not completely understood. This is critical to understanding the caveats of these new systems and screening for structural and functional artifacts that might be caused in the membrane proteins they are used to study. To better understand these potential effects, the fluid properties of dipalmitoylphosphatidylcholine lipid bilayers were examined by electron paramagnetic resonance (EPR) spectroscopy with spin-labeled reporter lipids in either liposomes or incorporated into nanoparticles with the copolymers diisobutylene-maleic acid or styrene maleic acid. Lineshape analysis at varying temperatures reveal a major change in the phase transition behavior of the lipids from a sharp melting curve in liposomes to a more gradual transition in nanoparticles. Electron spin echo envelope modulation (ESEEM) spectroscopy reveals changes in water permeability between mimetic systems, which is further supported by power-saturation measurements showing increased dequenching of spin lipids in diisobutylene-maleic acid nanoparticles compared to maleic acid nanoparticles. These results suggest that diisobutylene-maleic acid nanoparticles may have more physiological fluid properties than styrene-maleic acid nanoparticles when incorporated with saturated phospholipids.

As novel promising anticancer candidates, the piano-stool type complexes of ruthenium, [RuCl(η⁶-p-cymene)(N,S-L_n)]PF₆, K₁-₄, were synthesized from the reaction of the substituted benzo[b]thiophene based thiosemicarbazone ligands (L_1-4) with [{RuCl(η⁶-p-cymene)}₂(μ-Cl)₂]. All complexes were fully characterized using elemental analysis, and spectroscopic methods such as FT-IR and ¹H NMR. The molecular masses of the complexes were proved by MALDI-TOF analysis. Single crystal X-ray diffraction study was employed in the structural elucidation of complex K₁ which shows a distorted octahedral geometry around the Ru(II) ion. Furthermore, spectroscopic methods revealed that in all complexes the ligands are coordinated to the metal center in neutral thione form via N, S donors. In this study, the effect of all ligands, complexes and commercial drugs with a different concentration on the viability of OVCAR-3, A2780 and OSE cells were compared. In this comparison, the cytotoxicity of ruthenium (II) complexes on two ovarian cancer cell lines (human A2780 and human OVCAR-3) was evaluated. For this purpose, the resazurin assay was performed. Based on our studies, complex K₂ showed the highest toxicity against OVCAR-3 and A2780 cells. The cytotoxic effect of K₂ was found to be higher than that of the commercial anticancer agents Oxalpin and Carbodex, 1.8-34.7-fold for OVCAR-3 cells and 1.9-11.8-fold for A2780 cells, respectively. These results provide insight into the potential of ruthenium (II) complexes as a cytotoxic agent for the treatment of ovarian cancer, particularly for primary tumors.

The outer mitochondrial membrane protein known as mitoNEET was discovered when it was labeled by a photoaffinity derivative of the anti-diabetes medication, pioglitazone. The biological role for mitoNEET and its specific mechanism for achieving this remains an active subject for research. There is accumulating evidence suggesting that mitoNEET could be a component of mitochondrial FeS cofactor biogenesis. The protein was composed of an N-terminal membrane associated domain and a C-terminal domain oriented to the cytosol. The cytosolic domain was an iron-sulfur (2Fe-2S) metalloprotein with a rare 3Cys/1His coordination environment. It was previously reported that mitoNEET formed dimers that were remarkably sensitive to pH, likely a consequence of the protonation of the single His-iron ligand. The hypothesis pursued in the research reported here was that perhaps the dissociation of mitoNEET was also sensitive to the redox state of the iron sulfur cluster. To use native electrospray ionization mass spectrometry (ESI-MS) to monitor the reduction reaction ammonium dithionite was envisioned as the appropriate reagent to avoid sodium ion adduct formation from sodium dithionite. The preparation of ammonium dithionite was updated and the compound had the same properties as the sodium salt with redox dyes and the oxidized form of glutathione. The dissociation of mitoNEET treated with ammonium dithionite anaerobically was readily evident as ammonium dithionite was found to be compatible with redox chemistry evaluated by native ESI-MS.

Dangler sites protruding from a core metallocluster were introduced into the bioinorganic lexicon in 2000 by R.D. Britt and co-workers in an analysis of the tetramanganese oxygen-evolving cluster in photosystem II. In this perspective, we consider whether analogous dangler sites could participate in the mechanism of dinitrogen reduction by nitrogenase. Two possible roles for dynamic danglers in the active site FeMo cofactor are highlighted that might occur transiently during turnover. The first role for a dangler involves the S2B belt sulfur associated with displacement by carbon monoxide and other ligands, while the second dangler role could involve the entire cluster upon displacement of the His-  $\alpha$  442 side chain to the molybdenum by a free carboxyl group of the homocitrate ligand. To assess whether waters might be able to interact with the cofactor, a survey of small ligands (water and alkali metal ions) contacting [4Fe4S] clusters in synthetic compounds and proteins was conducted. This survey reveals a preference for these sites to pack over the centers of 2Fe2S rhombs. Waters are excluded from the S2B site in the resting state of nitrogenase, suggesting it is unlikely that water molecules coordinate to the FeMo cofactor during catalysis. While alkali metal ions are found to generally influence the properties of catalysts for dinitrogen reduction, no convincing evidence was found that any of the waters near the FeMo cofactor could instead be sodium or potassium ions. Dangler sites, if they exist in the nitrogenase mechanism, are likely formed transiently by localized changes to the resting-state FeMo cofactor structure.

The ferryl state in globins has previously been reported to undergo a protonation event below pH 5, as assessed using pH jump experiments with stopped-flow UV-Vis spectroscopy. This protonation entails hypsochromic shifts in the α and β bands (~ 20 to 40 nm) and an ~ 10 nm reduction in the energy difference between these two bands. We now report that in Mb this event is also characterized by a hypsochromic shift in the Soret band (~ 5 nm). No similar shifts in Soret, α, and β bands are seen upon the denaturation of ferryl Mb with guanidine-suggesting that the spectroscopic changes in ferryl Mb at pH < 5 are not caused by changes in the solvent exposure or in hydrogen bonding around the ferryl unit. Under the same denaturing conditions (pH jump below pH 5, and/or guanidine), ferric-aqua and ferrous-oxy Mb show no spectral changes of the order seen in the ferryl pH jump experiments. Together, these observations suggest that the protonation event is localized on the iron-bound oxygen atom, as opposed to somewhere on a hydrogen-bonding partner. Time-dependent density functional theory (TD-DFT) calculations were not able to systematically predict the UV-Vis spectra of the heme to the level of detail needed to interpret the experimental findings in this study.

Primary human neutrophils are the most abundant human white blood cells and are central for innate immunity. They act as early responders at inflammation sites, guided by chemotactic gradients to find infection or inflammation sites. Neutrophils can undergo both apoptosis as well as NETosis. NETosis is a form of neutrophil cell death that releases chromatin-based extracellular traps (NETs) to capture and neutralize pathogens. Understanding or controlling the balance between these cell-death mechanisms is crucial. In this study, the chemical synthesis and biologic assessment of a ruthenium complex as a light-activated photosensitizer that creates reactive oxygen species (ROS) in primary human neutrophils is reported. The ruthenium complex remains non-toxic in the dark. However, upon exposure to blue light at 450 nm, it exhibits potent cytotoxic effects in both cancerous and non-cancerous cell lines. Interestingly, the metal complex shifts the cell-death mechanism of primary human neutrophils from NETosis to apoptosis. Cells irradiated directly by the light source immediately undergo apoptosis, whereas those further away from the light source perform NETosis at a slower rate. This indicates that high ROS levels trigger apoptosis and lower ROS levels NETosis. The ability to control the type of cell death undergone in primary human neutrophils could have implications in managing acute and chronic infectious diseases.

The Mo-nitrogenase catalyzes the reduction of N₂ to NH₃ at the cofactor of its catalytic NifDK component. NifEN shares considerable homology with NifDK in primary sequence, tertiary structure and associated metallocenters. Better known for its biosynthetic function to convert an all-iron precursor (L-cluster; [Fe₈S₉C]) to a mature cofactor (M-cluster; [(R-homocitrate) MoFe₇S₉C]), NifEN also mimics NifDK in catalyzing substrate reduction at ambient conditions. The recently discovered ability of NifEN to reduce N₂ to NH₃ is particularly interesting, as it points to NifEN as a plausible, prototype ancient nitrogenase during evolution. Moreover, the dual function of NifEN in assembly and catalysis makes it a great template to reconstruct the functional variants or equivalents of NifDK, which could facilitate the mechanistic investigation and heterologous synthesis of nitrogenase. This perspective provides an overview of our recent studies of NifEN, with a focus on the implications of its functional versatility for nitrogenase assembly, catalysis and evolution.