Free energy contributions to cooperativity by the eight ligation intermediates of human hemoglobin (Hb) have been characterized extensively using six oxygenation analogs [cf. Huang et al. (1996) Biophys. J. 71, 2094-2105, Table 2]. These unprecedented data bses have strongly supported the molecular code mechanism of Hb cooperativity [Ackers et al. (1992) science 255, 54-83]. The present study addresses a recent argument against this work [Shibayama et al. (1997) Biochemistry 36, 4375-4381] based on "free energy" determinations for a doubly-ligated species of the CN-met analog. Shibayama et al. (1997) have claimed that, in the hybridization experiments that have been used to determine free energy of the asymmetric "species[21]" tetramer, a portion of the bound cyanide is allegedly released from CN-met Hb during the incubation with deoxy Hb that is used to achieve hybrid equilibrium. These authors have claimed that cyanide release has resulted in extensive electron exchange between heme sites of the hybridizing sample, leading to incorrect evaluation of the equilibrium species population by the cryogenic techniques that have been employed. In this report, we demonstrate that neither appreciable cyanide loss nor electron exchange occurs with the methods that have been used extensively by our two laboratories for these equilibrium determinations [Perrella et al. (1990) Biophys. Chem. 35, 97-103; Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. An alternative experiment, which Shibayama et al. (1997) have carried out to illustrate their claim, does not evaluate a thermodynamic equilibrium property of the species [21] hybrid. The relevance of their newly-estimated "free energy" is therefore unclear. Nevertheless, Shibayama et al. (1997) have claimed that their proposed "free energy" (which is approximately 1.3 kcal more positive than the free energy of -11.4 kcal found independently by our two laboratories) renders invalid the molecular code mechanism of hemoglobin cooperativity. This representation is utterly without foundation since a free energy even more positive than suggested by Shibayama et al. (1997) would be fully consistent with the molecular code mechanism.