Modulation of the pK_a of Metal-Bound Water via Oxidation of Thiolato Sulfur in Model Complexes of Co(lll)Containing Nitrile Hydratase:Insight into Possible Effect of Cysteine Oxidation in Co-Nitrile Hydratase

摘要

The Co(lll)complexes of N,N'-bis(2-mercaptophenyl)pyridine-2,6-dicarboxamide(PyPSH_4),a designed pentadentate ligand with built-in carboxamide and thiolate groups,have been synthesized and studied to gain insight into the role of Cys-S oxidation in Co-containing nitrile hydratase(Co-NHase).Reaction of[Co(NH_3)_5CI]Cl_2 with PyPS~(4-)in DMF affords the thiolato-bridged dimeric Co(lll)complex(Et_4N)_2[Co2(PyPS)_2](1).Although the bridged structure is quite robust,reaction of(Et_4N)(CN)with 1 in acetonitrile affords the monomeric species(Et_4N)_2[Co(PyPS)(CN)](2).Oxidation of 2 with H_2O_2 in acetonitrile gives rise to a mixture which,upon chromatographic purification,yields K_2[Co(PyPSO_2(OSO_2)(CN](3),a species containing asymmetrically oxidized thiolates.The Co(lll)metal center in 3 is coordinated to a S-bound sulfinate and an 0-bound sulfonate(OSO_2)group.Upon oxidation with H_2O_2,1 affords an asymmetrically oxidized dimer(Et_4N)_2[Co_2(PyPS(SO_2))_2](4)in which only the terminal thiolates are oxidized to form S-bound sulfinate groups while the bridging thiolates remain unchanged.The thiolato-bridge in 4 is also cleaved upon reaction with(EttNXCN)in acetonitrile,and one obtains(Et_4N)_2[Co(PyPS(SO_2))(CN)](5),a species that contains both coordinated thiolate and S-bound sulfinate around Co(lll).The structures of 1-4 have been determined.The spectroscopic properties and reactivity of all the complexes have been studied to understand the behavior of the Co(lll)site in Co-NHase.Unlike typical Co(lll)complexes with bound CN~-ligands,the Co(lll)centers in 2 and 5 are labile and rapidly lose CN~-in aqueous solutions.Since 3 does not show this lability,it appears that at least one thiolato sulfur donor is required in the first coordination sphere for the Co(lil)center in such species to exhibit lability.Both 2 and 5 are converted to the aqua complexes[Co(PyPS)(H_2O)]~-and[Co(PyPS(SO_2)(H_2O)]~ in aqueous solutions.The pK_a values of the bound water in these two species,determined by spectrophotometry,are 8.3(+-)0.03 and 7.2(+-)0.06,respectively.Oxidation of the thiolato sulfur(to sulfinate)therefore increases the acidity of the bound water.Since 2 and 5 promote hydrolysis of acetonitrile at pH values above their corresponding pK_a values,it is also evident that a metal-bound hydroxide is a key player in the mechanism of hydrolysis by these model complexes of Co-NHase.The required presence of a Cys-sulfinic residue and one water molecule at the Co(lll)site of Co-NHase as well as the optimal pH of the enzyme near 7 suggests that(i)modulation of the pKa of the bound water molecule at the active site of the enzyme could be one role of the oxidized Cys-S residue(s)and(ii)a cobalt-bound hydroxide could be responsible for the hydrolysis of nitriles by Co-NHase.