In order to gain an understanding of the role of the sulfenyl group of nitrile hydratase (NHase), a new Co(III) complex with a sulfenyl-type ligand (L-c=o:N-2(SO)(2)), Na[Co-III (L-c=o:N-2(SO)(2))(tBuNC)(2)] (2), was synthesized. The compound includes two amide groups, two sulfenate sulfurs in the equatorial plane, and two tBuNC molecules in the axial positions. Characterization of the compound was performed by UV-vis spectroscopic, IR spectral, thermogravimetric (TG), and X-ray structure analytical methods. The results are discussed in the context of Co(III) complexes containing the corresponding sulfur-type (L-c=o:N2S2) (1) and sulfinyl-type ligands (L-c=o:N-2(SO2)(2)) (3). Complex 2 crystallized with the formula Na[Co-III(L-c=o:N-2(SO)(2))(tBuNC)(2)]center dot urea-2H(2)O center dot 0.5EtOH. The X-ray structure revealed that the Co(III) complex has an octahedral geometry with Co-S = av. 2.221 (A) over circle, Co-N = av. 1.998 (A) over circle, and Co-C = av. 1.87 (A) over circle. The sulfenyl oxygen and amidate carbonyl oxygen are linked to urea, water, EtOH, and Na+ and participate in a hydrogen-bond and an electrostatic interaction. IR and TG measurements demonstrated that the coordination strength of tBuNC to the Cc atom increases as follows: 1 < 2 < 3. Complex 2 has almost the same stability as 3 in all solutions tested, although 1 exhibits a release of axial ligands in nonaqueous solutions. DFT calculations for 1, 2, and 3 demonstrated that Milliken atomic charges of the Co(III) centers are +1.466, +1.536, and +1.542, respectively, indicating that the extent of oxidation of the sulfur atoms increases the Lewis acidity of the Co(III) centers. Interestingly, the solution-state IR spectrum of 2 exhibits a solvent-dependent S-O stretching frequency. The frequency decreases with an increase in the electrophilicity (acceptor number) of the solvent. This solvent dependence was not observed for 3, which has a sulfinate (SO2) group, suggesting that the sulfenyl oxygen atom has nucleophilic character and promotes strong binding of the tBuNC molecule to lower the reaction barrier. These findings may suggest that the sulfenate oxygen in native NHase acts as a base (proton acceptor) and contributes to the activation of a water molecule and/or nitrile molecule.