Figure 4: Stack plot of substrate-induced and turnover-induced changes in the [Co^II]-EPR of PceA.

The red trace in the left-hand panel is the spectrum of the as-isolated enzyme. Subsequent traces are for incubation of the as-isolated PceA with 2,4,6-TBP (green trace), 2,4-DBP (blue trace), PCE (magenta trace), TCE (cyan trace), cis-1,2-DCE (black trace), TBE (violet trace) and cis/trans-1,2-dibromoethene (cis/trans-DBE, gold trace). In the right-hand panel, the red trace is the spectrum of reduced PceA. Reduction of PceA (3 mg ml⁻¹) with 1 mM Ti(III) citrate and 20 μM methyl viologen for about 5 min led to a [4Fe–4S]¹⁺ spectrum accompanied by the spectra of Ti(III) citrate and a methyl viologen radical. The lack of a [Co^II]-signal indicated the reduction of the cobalt to the EPR silent [Co^I]-state. The black dotted line is the spectrum of Ti(III) citrate alone. The subsequent traces are the equivalents of the traces in the left-hand panel after turnover of substrates induced by reduction of PceA for about 5 min. Experimental EPR conditions were: microwave frequency, 9,338 MHz; microwave power, 12.7 mW; modulation frequency, 100 kHz; modulation amplitude, 8 Gauss; temperature, 22 K.