Aging-Associated Enzyme Human Clock-1: Substrate-Mediated Reduction of the Diiron Center for 5‑Demethoxyubiquinone Hydroxylation

The mitochondrial membrane-bound enzyme Clock-1 (CLK-1) extends the average longevity of mice and <i>Caenorhabditis elegans</i>, as demonstrated for Δ<i>clk-1</i> constructs for both organisms. Such an apparent impact on aging and the presence of a carboxylate-bridged diiron center in the enzyme inspired this work. We expressed a soluble human CLK-1 (hCLK-1) fusion protein with an N-terminal immunoglobulin binding domain of protein G (GB1). Inclusion of the solubility tag allowed for thorough characterization of the carboxylate-bridged diiron active site of the resulting GB1-hCLK-1 by spectroscopic and kinetic methods. Both UV–visible and Mössbauer experiments provide unambiguous evidence that GB1-hCLK-1 functions as a 5-demethoxyubiquinone-hydroxylase, utilizing its carboxylate-bridged diiron center. The binding of DMQ_<i>n</i> (<i>n</i> = 0 or 2) to GB1-hCLK-1 mediates reduction of the diiron center by nicotinamide adenine dinucleotide (NADH) and initiates O₂ activation for subsequent DMQ hydroxylation. Deployment of DMQ to mediate reduction of the diiron center in GB1-hCLK-1 improves substrate specificity and diminishes consumption of NADH that is uncoupled from substrate oxidation. Both <i>V</i>_max and <i>k</i>_cat/<i>K</i>_M for DMQ hydroxylation increase when DMQ₀ is replaced by DMQ₂ as the substrate, which demonstrates that an isoprenoid side chain enhances enzymatic hydroxylation and improves catalytic efficiency

Decoding the Parkinson’s Symphony: PARIS, Maestro of Transcriptional Regulation and Metal Coordination for Dopamine Release

Parkin interacting substrate (PARIS) is a pivotal transcriptional regulator in the brain that orchestrates the activity of various enzymes through its intricate interactions with biomolecules, including nucleic acids. Notably, the binding of PARIS to insulin response sequences (IRSs) triggers a cascade of events that results in the functional loss in the substantia nigra, which impairs dopamine release and, subsequently, exacerbates the relentless neurodegeneration. Here, we report the details of the interactions of PARIS with IRSs via classical zinc finger (ZF) domains in PARIS, namely, PARIS(ZF2–4). Our biophysical studies with purified PARIS(ZF2–4) elucidated the binding partner of PARIS, which generates specific interactions with the IRS1 (5′-TATTTTT, Kd = 38.9 ± 2.4 nM) that is positioned in the promoter region of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). Mutational and metal-substitution studies demonstrated that Zn(II)–PARIS(ZF2–4) could recognize its binding partner selectively. Overall, our work provides submolecular details regarding PARIS and shows that it is a transcriptional factor that regulates dopamine release. Thus, PARIS could be a crucial target for therapeutic applications

CO₂ Enhanced Chemical Vapor Deposition Growth of Few-Layer Graphene over NiO_<i>x</i>

The use of mild oxidants in chemical vapor deposition (CVD) reactions has proven enormously useful. This was also true for the CVD growth of carbon nanotubes. As yet though, the use of mild oxidants in the CVD of graphene has remained unexplored. Here we explore the use of CO₂ as a mild oxidant during the growth of graphene over Ni with CH₄ as the feedstock. Both our experimental and theoretical findings provide in-depth insight into the growth mechanisms and point to the mild oxidants playing multiple roles. Mild oxidants lead to the formation of a suboxide in the Ni, which suppresses the bulk diffusion of C species suggesting a surface growth mechanism. Moreover, the formation of a suboxide leads to enhanced catalytic activity at the substrate surface, which allows reduced synthesis temperatures, even as low as 700 °C. Even at these low temperatures, the quality of the graphene is exceedingly high as indicated by a negligible D mode in the Raman spectra. These findings suggest the use of mild oxidants in the CVD fabrication as a whole could have a positive impact