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 Caenorhabditis elegans, as demonstrated for Δclk-1 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[subscript n] (n = 0 or 2) to GB1-hCLK-1 mediates reduction of the diiron center by nicotinamide adenine dinucleotide (NADH) and initiates O[subscript 2] 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 V[subscript max] and [k[subscript cat] over K[subscript M]] for DMQ hydroxylation increase when DMQ[subscript 0] is replaced by DMQ[subscript 2] as the substrate, which demonstrates that an isoprenoid side chain enhances enzymatic hydroxylation and improves catalytic efficiency.National Institute of General Medical Sciences (U.S.) (Grant GM032134)National Science Council (China). Postdoctoral Research Abroad ProgramAlexander von Humboldt-Stiftung (Fellowship