Electron transfer within xanthine oxidase: A solvent kinetic isotope effect study
- Ohio State Univ., Columbus (United States)
Solvent kinetic isotope effect studies of electron transfer within xanthine oxidase have been performed, using a stopped-flow pH-jump technique to perturb the distribution of reducing equivalents within partially reduced enzyme and follow the kinetics of reequilibration spectrophotometrically. It is found that the rate constant for electron transfer between the flavin and one of the iron-sulfur centers of the enzyme observed when the pH is jumped from 10 to 6 decreases from 173 to 25 s{sup {minus}1} on going from HJ{sub 2}O to D{sub 2}O, giving an observed solvent kinetic isotope effect of 6.9. An effect of comparable magnitude is observed for the pH jump in the opposite direction, the rate constant decreasing form 395 to 56 s{sup {minus}1}. The solvent kinetic isotope effect on k{sub obs} is found to be directly proportional to the mole fraction of D{sub 2}O in the reaction mix for the pH jump in each direction, consistent with the effect arising from a single exchangeable proton. Calculations of the microscopic rate constants for electron transfer between the flavin and the iron-sulfur center indicate that the intrinsic solvent kinetic isotope effect for electron transfer from the neutral flavin semiquinone to the iron-sulfur center designated Fe/S I is substantially greater than for electron transfer in the opposite direction and that the observed solvent kinetic isotope effect is a weighted average of the intrinsic isotope effects for the forward and reverse microscopic electron-transfer steps. The results emphasize the importance of prototropic equilibria in the kinetic as well as thermodynamic behavior of xanthine oxidase and indicate that protonation/deprotonation of the isoalloxazine ring is concomitant with electron transfer in the xanthine oxidase system.
- OSTI ID:
- 5821306
- Journal Information:
- Biochemistry; (United States), Vol. 30:35; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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HEAVY WATER
ISOTOPE EFFECTS
OXIDOREDUCTASES
BIOCHEMICAL REACTION KINETICS
ELECTRON TRANSFER
FLAVINES
THERMODYNAMIC ACTIVITY
XANTHINES
ACRIDINES
AMINES
AROMATICS
AZAARENES
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ENZYMES
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
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ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
OXYGEN COMPOUNDS
PROTEINS
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550201* - Biochemistry- Tracer Techniques