The active site A-cluster in the a subunit of the title enzyme consists of an Fe4S4 cluster coordinated to a [Ni-p Ni-d] subcomponent, The cluster must be activated for catalysis using low-potential reductants such as Ti(III) citrate. Relative to the inactive {[Fe4S4)(2+) Ni-p(2+) Ni-d(2+)} state, the activated state appears to be 2-electrons more reduced, but the location of these electrons within the A-cluster is uncertain, with {[Fe4S4](2+) Ni-p(2+) Ni-d(2+)} and {[Fe4S4](1+) Ni-p(1+) Ni-d(2+)) configurations proposed. Recombinant apo-alpha subunits oligomerize after activation with NiCl2. The dimer fraction, upon reduction with excess Ti(III)citrate, exhibited Mossbauer spectra consisting of two quadrupole doublets representing 51% and 21% of the Fe, with parameters indicating [Fe4S4](1+) states. Spectra recorded in strong magnetic fields were typical of diamagnetic systems, indicating an exchange-coupled S = 0 {[Fe4S4](1+) Ni-p(1+)) state. Additional treatment with CO altered the doublet Mossbauer parameters, suggesting an interaction with CO, but maintaining the cluster in the {[Fe4S4](1+) Ni-p(1+)) state. Reduction with substoichiometric equivalents of Ti(III) citrate afforded an EPR signal typical of Ni1+ ions, with g. = 2.10 and g(perpendicular to) = 2.02. Addition of more Ti caused the signal intensity to decline, suggesting that it arises from the semireduced {[Fe4S4](2+) Nip(1+)} state.