Structural and mechanistic insights into the sulfur transfer protein SufU from Staphylococcus aureus.

Substitution of the active site Zn2+ ion in the sulfur transfer protein (SufU) from Staphylococcus aureus (SaSufU) with a catalytically active Co2+ ion revealed a S-➔Co2+ ligand-to-metal-charge-transfer (LMCT) band at 340 nm with an ε of ∼2,760M-1cm-1, as well as d-d (i.e. ligand field) absorption bands at 584 nm (ε = 540 ), 636 nm (ε = 580 ) and 735 nm (ε = 220 ). These data suggest a distorted four- or five-coordinate Co2+ center ligated by 3 Cys residues with a dissociation constant (Kd)of ∼600nM. Co K-edge absorption spectra (XAS) of Co2+-SaSufU indicated five-coordinate Co2+, while extended X-ray absorption fine structure (EXAFS) spectra of Co2+-SaSufU revealed 2 N/O and 3 S ligands. EXAFS data for Zn2+-SaSufU revealed 1 N/O and 3 S ligands consistent with the proposed Cys3Asp coordinating residues. The fifth ligand in the Co2+-SaSufU enzyme is either a bidentate the active site Asp ligand or an exogenous water. Upon the addition of SaSufS to either Zn2+-SaSufU or Co2+-SaSufU, XAS and EXAFS data suggest that one of the S ligands is displaced by an N/O atom donor. Interestingly, electronic absorption data suggest that this only occurs in the presence of Cys. EPR analysis of Co2+-SaSufU revealed temperature- and B1-dependent transitions within both the Ms=±12 and MS=±32 doublets of the S=32 spin system that suggested either heterogeneity of the electronic structure or an unusually small zero-field splitting for Co(II). Taken together, these data provide in-solution evidence for the mechanism of S2- transfer from SaSufS to SaSufU.