Cobalt-substituted Fe-type nitrile hydratase of Rhodococcus sp N-771

When the genes encoding alpha and beta subunits of Fe-type nitrile hydratas e (NHase) from Rhodococcus sp. N-771 were expressed in Escherichia coli in Go-supplemented medium without co-expression of the NHase activator, the NH ase specifically incorporated not Fe but Co ion into the catalytic center. The produced Go-substituted enzyme exhibited rather weak NHase activity ini tially. However, the activity gradually increased by the incubation with an oxidizing agent, potassium hexacyanoferrate. The oxidizing agent is likely to activate the Co-substituent by oxidizing the Co atom to a low-spin Co3 State and/or modification of alpha Cys-112 to a cysteine-sulfinic acid. It is suggested that the NHase activator not only supports the insertion of a n Fe ion into the NHase protein but also activates the enzyme via the oxida tion of its iron center. (C) 2000 Federation of European Biochemical Societ ies.