Density functional theory calculations on the active site of biotin synthase: mechanism of S transfer from the Fe2S2 cluster and the role of 1st and 2nd sphere residues

Density functional theory (DFT) calculations are performed on the active site of biotin synthase (BS) to investigate the sulfur transfer from the Fe2S2 cluster to dethiobiotin (DTB). The active site is modeled to include both the 1st and 2nd sphere residues. Molecular orbital theory considerations and calculation on smaller models indicate that only an S atom (not S2-) transfer from an oxidized Fe2S2 cluster leads to the formation of biotin from the DTB using two adenosyl radicals generated from S-adenosyl-l-methionine. The calculations on larger protein active site model indicate that a 9-monothiobiotin bound reduced cluster should be an intermediate during the S atom insertion from the Fe2S2 cluster consistent with experimental data. The Arg260 bound to Fe-1, being a weaker donor than cysteine bound to Fe-2, determines the geometry and the electronic structure of this intermediate. The formation of this intermediate containing the C9-S bond is estimated to have a Delta G(not equal) of 17.1 kcal/mol while its decay by the formation of the 2nd C6-S bond is calculated to have a Delta G(not equal) of 29.8 kcal/mol, i.e. the 2nd C-S bond formation is calculated to be the rate determining step in the cycle and it leads to the decay of the Fe2S2 cluster. Significant configuration interaction (CI), present in these transition states, helps lower the barrier of these reactions by similar to 30-25 kcal/mol relative to a hypothetical outer-sphere reaction. The conserved Phe285 residue near the Fe2S2 active site determines the stereo selectivity at the C6 center of this radical coupling reaction.