The reactivity of chicken liver xanthine dehydrogenase with molecular oxygen.
The reaction of 6-electron reduced chicken liver xanthine dehydrogenase (XDH) with molecular oxygen was studied using both stopped flow and steady-state turnover techniques at pH 7.8, 4 degrees C. Oxidation of fully reduced XDH proceeded via four phases, three of which were detected with the stopped flow spectrophotometer. The fastest phase was second order in oxygen (1900 M-1 s-1), resulted in the appearance of flavin semiquinone and yielded no superoxide. The next phase was also second order in oxygen (260 M-1 s-1), involved the loss of flavin semiquinone and yielded, on average, 1 mol of superoxide/mol of XDH oxidized. The last 2 electron equivalents were located in the iron-sulfur centers. They were released one equivalent at a time in the form of superoxide. Steady-state kinetics were found to be critically dependent on temperature and oxygen concentration. When these factors were carefully controlled, both the xanthine-oxygen and NADH-oxygen reductase reactions gave linear Lineweaver-Burk plots. The xanthine-oxygen data yielded a turnover number of 43 min-1, which was 42% of that for xanthine-NAD turnover. During turnover, with xanthine and O2, 40-44% of the electron equivalents introduced by xanthine appeared as superoxide. Reduced pyridine nucleotides, NAD and 3-aminopyridine adenine dinucleotide, dramatically reduced the formation of superoxide at levels which did not seriously inhibit oxygen reactivity.
Nishino
T
T
Department of Biochemistry, Yokohama City University School of Medicine, Japan.
Nishino
T
T
Schopfer
L M
LM
Massey
V
V
D000332:Aerobiosis; D000693:Anaerobiosis; D000818:Animals; D002645:Chickens; D007658:Ketone Oxidoreductases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism*; D013053:Spectrophotometry; D014968:Xanthine Dehydrogenase / Q000378:metabolism*
D014968:Xanthine Dehydrogenase; D007658:Ketone Oxidoreductases; D010100:Oxygen