Mossbauer and EPR study of the Ni-activated alpha-subunit of carbon monoxide dehydrogenase from Clostridium thermoaceticum
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The A-center of carbon monoxide dehydrogenase (CODH) resides in the enzyme's -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 -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 = 1/4 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/. In order to elucidate the structure of the A-cluster and to understand the cause of the substoichiometric spin intensities, we have studied the -subunit with Mossbauer and EPR spectroscopy. As found for CODH, populations of isolated 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 exhibit Mossbauer spectra (S = 0, E(Q) = 1.08 mm/s, = 0.45 mm/s) typical of [Fe4S4]2+ clusters. Upon reduction with dithionite, the Fe4S4 cluster of nonlabile A-clusters exhibits Mossbauer (average E(Q) = 1.0 mm/s, 0.54 mm/s) and EPR properties similar to those of S = 3/2 [Fe4S4]+ 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 Fe4S4 portion of the A-cluster exhibits 57Fe 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 E(Q) and show that the cluster has remained in the [Fe4S4]2+ state. We explain these observations with an electronic model that considers a Ni+-X-[Fe4S4]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) = jS(Ni)S(Fe)). The Mossbauer spectre provide no evidence that CO is bound to the Fe4S4 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 Ni2+ must be low-spin (S(Ni) = 0).
