EPR spectra of (a) the one-CO-bound (lo-CO) state generated in the presence of 1 atm CO (1, black trace) and (b) the multi-CO-bound (hi-CO) state generated in the presence of 2.6 atm CO (1, dark green trace). Both states were generated with the resting-state VnfDGKAv (i.e., without the Fe protein, the reductase partner of VnfDGKAv) using EuII-DTPA as a reductant. (c) The difference spectrum between the hi-CO (b, 1) and lo-CO state (a, 1) reflects features generated upon binding of ‘extra’ CO molecules (1, gray trace). The g values are indicated in the figure. Spectra (a, 1), (b, 1) and (c, 1) were simulated as described in the Experimental Section and are shown as traces (a, 2), (b, 2) and (c, 2) below the corresponding spectra. (d) Quantification of the amounts of VnfDGKAv-bound CO in the lo-CO (3) and hi-CO (4) states. The amount of bound CO was quantified upon acid quench of the hi- or lo-CO states of VnfDGKAv. The same experiment was conducted on the V-cluster-deficient, apo VnfDGKAv, which was incapable of capturing CO under the conditions described for the formation of the lo-CO (1) and hi-CO (2) states.