Single turnover EPR studies of benzoyl-CoA reductase

Benzoyl-CoA reductase (BCR) catalyzes the ATP-driven transport of two elect rons from a reduced 2[4Fe-4S] ferredoxin to the aromatic ring of benzoyl-Co A. A mechanism involving radical species and very low potential electrons s imilar to the Birch reduction of aromatics has been suggested for this reac tion, The redox centers of BCR have previously been identified, by EPR- and Mossbauer spectroscopy, to be three cysteine-ligated [4Fe-4S] clusters [Bo ll et al. (2000) J. Biol. Chem. 275, 31857-31868] with redox potentials mor e negative than -500 mV, In this work, the catalytic cycle of BCR was studi ed by freeze-quench experiments; the dithionite reduced, enzyme was rapidly mixed with equimolar amounts of benzoyl-CoA and excess MgATP plus dithioni te, 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 concentra tion increased 3-fold very rapidly (within similar to 25 ms). In the course of a single turnover the extent of enzyme reduction decreased again, final ly reaching the starting value. An increased magnetic interaction of [4Fe-4 S] clusters and the rise of an S = 7/2 high-spin EPR signal occurred as sec ond simultaneous and transient events (at similar to 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 t o high-spin EPR signals. Finally, two novel transient EPR signals with an i sotropic line-shape developed maximally in the late phase of the catalytic cycle (similar to1-2 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 spec ies with [4Fe-4S](+1) clusters, On the basis of these results, we present a proposal for a catalytic cycle involving radical species.