The insertion of a second disulfide bridge into native pig heart lipoamide dehydrogenase, requires two Cu-2+ ions for each catalytic center inactivated under anaerobic conditions. During inactivation, both metal atoms become reducible by their juxtaposition to the two participating cysteine residues and may be removed as the Cu+-chelates of neocuproine and bathocuproinesulfonate, leaving an additional disulfide bridge on the protein. Inactivation does not require the presence of oxygen, but when substoichiometric levels of copper are used under aerobic conditions the slow regeneration of Cu-2+ becomes rate-limiting. The course of aerobic inactivation is markedly biphasic at 0 degrees using 2 Cu-2+/FAD, with 30% of the total change completed rapidly, followed by a much slower phase. Both the extent of the fast phase and the rate of the second phase are enhanced by increasing levels of Cu-2+, but are relatively unaffected when the Cu-2+/FAD ratio is maintained at 2 and the protein concentration is varied. The enzyme affords several binding sites for Cu-2+ at pH 7.8, and it is suggested that competition between these sites during the initial statistical distribution of metal ions may explain this biphasic behavior.