Metal binding to D-lactate dehydrogenase

The kinetic and spectral properties of native and cobalt-substituted D-lactate dehydrogenase have been compared. Optical and MCD spectra are consistent with high-spin tetrahedral cobalt. The cobalt enzyme is strongly inhibited by D-lactate when oxygen is the electron acceptor. This is probably due to the formation of an abortive complex of reduced enzyme and lactate. Like the native zinc enzyme, the cobalt-substituted enzyme is reduced by substrate in a triphasic fashion involving a transient charge-transfer complex. The kinetics of the cobalt enzyme suggest that the metal is involved in substrate binding and reduction. A second metal-binding site is also described. Evidence is presented to suggest that the flavin cofactor and an essential thiol are involved in this site.