tailieunhanh - Báo cáo khoa học: The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase

The influence of temperature on cytochromec oxidase (CCO) catalytic activity was studied in the temperature range 240–308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcalÆmol )1 ; the molecular activity or turnover number was 12 s )1 at 280K in the absence of ethylene glycol. CCO activity was studied between 240and 277 K in the presence of ethylene glycol. | Eur. J. Biochem. 270 253-260 2003 FEBS 2003 doi The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase Jack A. Kornblatt1 Bruce C. Hill2 and Michael C. Marden3 1 Enzyme Research Group Concordia University Montreal Quebec Canada 2Department of Biochemistry Queen s University Kingston Ontario Canada 3INSERM U473 Le Kremlin-Bicetre Cedex France The influence of temperature on cytochrome c oxidase CCO catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal-mol-1 the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CO activity was studied between 240and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal-mol-1 the molecular activity was 1 s-1 at 280 K. Ethyeene glyool inhibits CCO by lowering the activity of water. The rate limitation in electron transfer ET was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1 150 s-1 and 30 s-1 and these dropped to 220 s-1 27 s-1 and 3 s-1 at 240 K. The atií-vation energies were 5 kcal-mol-1 7 kcal-mol-1 and 8 kcal-mol-1 respectively. The fastest rate we ascribe to the oxidation of cytochrome a3 the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important a with vs. without ethylene glycol and b steady state vs. flash-induced oxidation. When one makes these two comparisons it is