Redox potentials of milk xanthine dehydrogenase. Room temperature measurement of the FAD and 2Fe/2S center potentials.
Xanthine oxidase (XO) and xanthine dehydrogenase (XDH), two forms of the same enzyme isolated from cow's milk, have differing redox potentials of their chromophores. Both XDH and XO are capable of accepting 8 electrons per active site cluster of redox acceptors. By titrating XDH with redox indicator dyes of various potentials, the potentials have been determined for the flavin as well as for the 2Fe/2S centers of the enzyme at pH 7.5, 25 degrees C. The redox potential for the FAD/FADH. half-potential was found to be -270 +/- 5 mV and that for the FADH./FADH2 half potential, -410 +/- 5 mV. The first flavin half potential is close to the value which has been reported for XO (Porras, A. G., and Palmer, G. (1982) J. Biol. Chem. 257, 11617-11626). However, the second FAD half-potential is 180 mV lower in XDH than in XO, creating a 140-mV separation between the FAD potentials in XDH. This separation gives rise to a maximum development of the flavin semiquinone in XDH near 0.9 equivalent as confirmed by EPR quantitation of FADH. formed during reductive titrations. The potentials of both the 2Fe/2S centers in XDH were determined and found to be identical to the values which were found for the iron-sulfur centers in XO.
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
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J
Hunt
V
V
Massey
W R
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Dunham
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Sands
D000818:Animals; D001665:Binding Sites; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008892:Milk / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism; D010619:Phenazines; D010777:Photochemistry; D013053:Spectrophotometry; D014968:Xanthine Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
D007461:Iodoacetates; D007506:Iron-Sulfur Proteins; D010619:Phenazines; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D004227:Dithionite; D014968:Xanthine Dehydrogenase; D010269:Paraquat; D019807:Iodoacetic Acid; C009195:safranine T