Kinetic studies (25-degrees-C) on the transformation Fe3S40 + M2+ reversible Fe3MS42+, in which M2+ enters the vacant subsite of an [3Fe-4S] incomplete cuboidal structure of aconitase to give Fe3MS42+ products, are reported. The studies described with M = Fe are part of the enzyme reactivation process, and the product has been confirmed as the Fe4S42+ cluster. With all three metal ions M = Fe, Mn, and Co, uniphasic equilibration processes are observed, yielding at pH 7.4 formation rate constants k1/M-1 s-1 of 1.9 (Fe2+), 1.0 (Mn2+), and 5.9 (Co2+). Together with rate constants (k2) for the reverse processes, equilibrium constants 10(-2) K/M-1 for incorporation of 6.1 (Fe2+), 2.2 (Mn2+), and 13.5 (Co2+) are obtained. In the case of [Co2+] > 1.5 X 10(-3) M, a second phase corresponding to a denaturation process has to be allowed for, and with Ni2+, the latter becomes the dominant process, preventing any determination of rate Constants. Values of k1 are > 105 times smaller than rate constants for H2O exchange on M2+ hexaaqua ions, reflecting in part the inaccessibility of the Fe3S40 Cluster. Aconitase is a basic protein (pI approximately 8.5), and the overall electrostatics for reaction with M2+ are also unfavorable. More favorable is the reaction with Fe(H2O)5SO4 formed on addition of SO42-, which reacts 140 times faster than hexaaqua Fe2+. The K values obtained together with those from electrochemical studies on Fe2+, Zn2+, and Cd2+ incorporation into the Desulfovibrio africanus Fe3S40 cluster are in the order Cd2+ > Zn2+ > Co2+ > Fe2+ > Mn2+. This order is as expected for the coordination of 2+ metal ions to electron-rich sulfido groups.