ARCHIVES
OF
BIOCHEMISTRY
Kinetics
AND
BIOPHYSICS
182, 107-117 (1977)
of the Reoxidation
of Succinate
Dehydrogenasel
BRIAN A. C. ACKRELL,” EDNA B. KEARNEY,* C. J. COLES,” THOMAS P. SINGER,*, 2 HELMUT BEINERT,t YIEH-PING WAN,+ AND KARL FOLKERSS *Molecular Biology Division, Veterans Administration Hospital, San Francisco, California 94121, and the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143; ‘tznstitute of Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706; and $Znstitute for Biomedical Research, University of Texas, Austin, Texas 78712 Received January
21, 1977
The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations’ by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q1 analog, 2,3-dimethoxy-5methyl-6-pentyl-1,4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only -80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previovsly in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in’
OF
BIOCHEMISTRY
Kinetics
AND
BIOPHYSICS
182, 107-117 (1977)
of the Reoxidation
of Succinate
Dehydrogenasel
BRIAN A. C. ACKRELL,” EDNA B. KEARNEY,* C. J. COLES,” THOMAS P. SINGER,*, 2 HELMUT BEINERT,t YIEH-PING WAN,+ AND KARL FOLKERSS *Molecular Biology Division, Veterans Administration Hospital, San Francisco, California 94121, and the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143; ‘tznstitute of Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706; and $Znstitute for Biomedical Research, University of Texas, Austin, Texas 78712 Received January
21, 1977
The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations’ by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q1 analog, 2,3-dimethoxy-5methyl-6-pentyl-1,4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only -80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previovsly in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in’
