Abstract: Objective To explore the mechanisms of transcription factor circadian locomalor output cycles kaput(CLOCK) in mediating regulatory function of endogenous hydrogen peroxide(H₂O₂) for cell respiration. Methods Cellular oxygen consumption capacity and glycolysis in Clock^C195S mouse embryonic fibroblasts (MEFs) and adult fibroblasts(MAFs) were tested by Agilent Seahorse XF Cell Mito Stress Test Kit and Glycolysis Stress Test Kit. The expression of key genes participating in cell respiration was detected by q-PCR. The endogenous H₂O₂ levels and the redox modification of CLOCK were detected by Amplex^® Red Hydrogen Peroxide/Peroxidase Assay Kit and biotin-conjugated iodoacetamide (BIAM) were used respectively after the treatment of antioxidant Trolox. Changes in the oxygen consumption capacity and in key respiratory gene expression after the treatment of antioxidant Trolox were also tested in Clock^wt and Clock^C195S MEFs. Results Cellular oxygen consumption capacity, glycolysis and the expression of NMNAT2, a key enzyme in nicotinamide adenine denudeotide(NAD) biosynthesis, as well as NAD content all decreased in Clock^C195S MEFs. The treatment with Trolox decreased endogenous H₂O₂ level and dampened respiration capacity in Clock^wt MEFs but not in Clock^C195S MEFs. Conclusions CLOCK mediates the regulation of endogenous H₂O₂ for cellular respiration through its redox modification at Cys195.

Key words: cellular respiration, nicotinamide adenine denudeotide(NAD) biosynthesis, redox modification of CLOCK