Magnetic circular dichroism studies of succinate dehydrogenase. Evidence for [2Fe-2S], [3Fe-xS], and [4Fe-4S] centers in reconstitutively active enzyme.
Reconstitutively active and inactive succinate dehydrogenase have been investigated by low temperature magnetic circular dichroism (MCD) and EPR spectroscopy and room temperature CD and absorption spectroscopy. Reconstitutively active succinate dehydrogenase is found to contain three spectroscopically distinct Fe-S clusters: S1, S2, and S3. In agreement with previous studies, MCD and CD spectroscopy confirm that center S1 is a succinate-reducible [2Fe-2S]2+,1+ center. The MCD characteristics of center S2 identify it as a dithionite-reducible [4Fe-4S]2+,1+ similar to those in bacterial ferredoxins. EPR power saturation studies and the weakness of the EPR signal from reduced S2 indicate that there is a weak magnetic interaction between centers S1 and S2 in their paramagnetic, S = 1/2, reduced states. Center S3 is identified both by the form of the MCD spectrum and the characteristic magnetization behavior as a reduced [3Fe-xS] center in both succinate- and dithionite-reduced reconstitutively active succinate dehydrogenase. Arguments are presented in favor of centers S2 and S3 being separate centers rather than interconversion products of the same cluster. Reconstitutively inactive succinate dehydrogenase is found to be deficient in center S3. These results resolve many of the controversies concerning the Fe-S cluster content of succinate dehydrogenase and reconcile published EPR data with analytical and core extrusion studies. Moreover, they indicate that center S3 is a necessary requirement for reconstitutive activity and suggest that it is able to sustain ubiquinone reductase activity as a [3Fe-xS] center.
Johnson
M K
MK
Morningstar
J E
JE
Bennett
D E
DE
Ackrell
B A
BA
Kearney
E B
EB
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism / Q000379:methods; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008280:Magnetics; D008667:Metalloproteins / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013385:Succinate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D004227:Dithionite; D013385:Succinate Dehydrogenase