Copper in the cytosol of the hepatopancreas of the American lobster, Homarus americanus, occurs as copper-metallothionein [Cu(I)-MT] and as a copper-glutathione complex [Cu(I)-GSH]. The latter can act in vitro as the source of Cu(I) in the reconstitution of lobster apohemocyanin, whereas Cu(I)-MT cannot. Here we report on the mechanism of the GSH-mediated reconstitution. Binding of Cu(I) to apohemocyanin was measured by its effect on the protein's fluorescence, by ultrafiltration experiments and size-exclusion HPLC. Reconstitution of CO and O2 binding was studied using the [Cu(I)...Cu(I)-CO] fluorescence of hemocyanin and its Cu-O2-Cu charge-transfer band as spectral probes. The hemocyanin oligomer has 1 (1.02 +/- 0.09) high-affinity (apparent Kdiss = 1.67 +/- 0.40 microM) external binding site for ionic Cu(I) per subunit. Binding of Cu(I) to this site is fast and reversible and is followed by a slow, irreversible incorporation of copper into the protein matrix. Movement of the first copper through the matrix to the active site is the rate-limiting step in the reconstitution process. Mononuclear copper sites, once formed, are rapidly converted into biologically active, binuclear copper sites. In accordance with this reaction sequence, the restoration of CO/O2 binding by hemocyanin is a first-order reaction with a half-time of 100 +/- 5 min at pH 6.0. Reconstitution is extremely pH-dependent and proceeds best at those pH values where the architecture of the copper pocket of hemocyanin is open as judged from its extremely low affinity for oxygen and its very fast oxygen dissociation rate.(ABSTRACT TRUNCATED AT 250 WORDS)