title: Single turnover EPR studies of benzoyl-CoA reductase.
abstract: Benzoyl-CoA reductase (BCR) catalyzes the ATP-driven transport of two electrons from a reduced 2[4Fe-4S] ferredoxin to the aromatic ring of benzoyl-CoA. A mechanism involving radical species and very low potential electrons similar to the Birch reduction of aromatics has been suggested for this reaction. The redox centers of BCR have previously been identified, by EPR- and Mössbauer spectroscopy, to be three cysteine-ligated [4Fe-4S] clusters [Boll et al. (2000) J. Biol. Chem. 275, 31857-31868] with redox potentials more negative than -500 mV. In this work, the catalytic cycle of BCR was studied by freeze-quench experiments; the dithionite reduced enzyme was rapidly mixed with equimolar amounts of benzoyl-CoA and excess MgATP plus dithionite, and subjected to EPR spectroscopic analysis. The turnover period of the enzyme under the conditions used was 3 s. The total S = (1)/(2) spin concentration increased 3-fold very rapidly (within approximately 25 ms). In the course of a single turnover the extent of enzyme reduction decreased again, finally reaching the starting value. An increased magnetic interaction of [4Fe-4S] clusters and the rise of an S = (7)/(2) high-spin EPR signal occurred as second simultaneous and transient events (at approximately 200 ms). Previous work showed that binding of the nucleotide affects the magnetic interaction of [4Fe-4S] clusters, whereas hydrolysis of MgATP is required for the switch to high-spin EPR signals. Finally, two novel transient EPR signals with an isotropic line-shape developed maximally in the late phase of the catalytic cycle ( approximately 1-2 s). These signals differed from those of typical free radicals by shifted g values at g = 2.015 and g = 2.033 and by an unusually fast relaxation rate, suggesting an interaction of these paramagnetic species with [4Fe-4S](+1) clusters. On the basis of these results, we present a proposal for a catalytic cycle involving radical species.
authors: Institut für Biologie II, Universitat Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany. boll@uni-freiburg.de
authors: M
authors: M
authors: Boll
authors: G
authors: G
authors: Fuchs
authors: D J
authors: DJ
authors: Lowe
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D004227:Dithionite / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004579:Electron Transport; D005609:Free Radicals / Q000378:metabolism; D005615:Freezing; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013379:Substrate Specificity; D020628:Thauera / Q000201:enzymology
chemical_list: D000214:Acyl Coenzyme A; D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D004227:Dithionite; C037817:benzoyl-coenzyme A; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C109030:benzoyl-coenzyme A reductase (dearomatizing)