Mössbauer and EPR Study of the Ni-Activated α-Subunit of Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

The A-center of carbon monoxide dehydrogenase (CODH) resides in the enzyme's α-subunit and is responsible for the acetyl-CoA synthase activity. The center comprises a Ni site and an iron−sulfur cluster. We have isolated the α-subunit using both continuous and discontinuous electrophoresis methods. When incubated with CO, samples prepared using continuous gels attain the A red -CO state that exhibits an S = 1 / 2 EPR feature ( g = 2.048, 2.046, 2.021) similar to the so-called NiFeC signal of native CODH. Both signals consistently quantify to <0.4 spin/α. In order to elucidate the structure of the A-cluster and to understand the cause of the substoichiometric spin intensities, we have studied the α-subunit with Mössbauer and EPR spectroscopy. As found for CODH, populations of isolated α are heterogeneous: they contain two major A-cluster forms designated nonlabile and Ni-labile. In native CODH, only the Ni-labile form develops the NiFeC signal and exhibits catalytic activity. Oxidized samples of α exhibit Mössbauer spectra ( S = 0, Δ E Q = 1.08 mm/s, δ = 0.45 mm/s) typical of [Fe 4 S 4 ] 2+ clusters. Upon reduction with dithionite, the Fe 4 S 4 cluster of nonlabile A-clusters exhibits Mössbauer (average Δ E Q = 1.0 mm/s, δ = 0.54 mm/s) and EPR properties similar to those of S = 3 / 2 [Fe 4 S 4 ] + cubanes; in contrast, Ni-labile clusters are not reducible by dithionite. Treatment with CO yielded a sample for which 40% of the Fe was associated with A red -CO, while 47% of the clusters (the nonlabile form) remained oxidized. Thus, the presence of nonlabile A-clusters is largely responsible for the low spin intensities of the NiFeC signal. Upon formation of A red -CO, the Fe 4 S 4 portion of the A-cluster exhibits 57 Fe magnetic hyperfine interactions; the cluster sites divide into equivalent pairs with (isotropic) A A = A B = −34.2 MHz and A C = A D = +26.8 MHz. However, the values of Δ E Q and δ show that the cluster has remained in the [Fe 4 S 4 ] 2+ state. We explain these observations with an electronic model that considers a Ni + −X−[Fe 4 S 4 ] 2+ assembly for which the Ni + is exchange-coupled with one Fe site of the cube through a bridging ligand (X). The coupling was found to be substantial, namely | j | ≈ 100 cm -1 (ℋ NiFe = j S Ni · S Fe ). The Mössbauer spectra provide no evidence that CO is bound to the Fe 4 S 4 cluster in the state A red -CO, as has been concluded from resonance Raman studies [Qiu, D.; Kumar, M.; Ragsdale, S. W.; Spiro, T. G. Science 1994, 264, 817−819]. We could not determine if the two metal centers are linked in the oxidized state (for either the Ni-labile or nonlabile form), but if they are, the Ni 2+ must be low-spin ( S Ni = 0).