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

Abstract

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