ASYMMETRIC CYANOMET VALENCY HYBRID HEMOGLOBIN, (ALPHA(-)BETA(+CN-))(ALPHA-BETA) - THE ISSUE OF VALENCY EXCHANGE(CN)

A new framework for hemoglobin cooperativity was proposed by Ackers an d colleagues on the basis of the hyper thermodynamic stability and deo xy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha(+CN-)beta(+CN-))(alpha beta), studied by hybridiz ation of the equimolar mixture of deoxyhemoglobin and cyanomethemoglob in through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad, Sci. U.S.A. 88, 1110-1114]. Recently, we rep orted that the published hyperstability of (alpha(+CN-)beta(+CN-))(alp ha beta) is incorrect due to the occurrence of valency exchange betwee n the heme sites of both parental hemoglobins during the long deoxy in cubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free c yanide of the sample during the long incubation, which led to formatio n of cyanide-unbound aqometheme in the original deoxyhemoglobin result ing from the electron transfer to cyanometheme. This paper is a respon se to a recent argument against our work [Ackers et al. (1997) Biochem istry 36 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange m ay have occurred via aqometheme. In this paper, however, we demonstrat e that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are m aintained by a continuous flow of humidified N-2 With HCN. This confir ms our view that previous experimental data on (alpha(+CN-)beta(+CN-)) (alpha beta) Obtained by the long incubations should be subject to ree xamination while our earlier estimation of a lower limit of free energ y of (alpha(+CN-)beta(+CN-))(alpha beta) (i.e., greater than or equal to -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha(+CN-)b eta(+CN-))(alpha beta) assigned by Ackers and colleagues using the lon g incubations is an artifact arising from the valency exchange, These results suggest that the putative mechanistic picture for hemoglobin c ooperativity inferred from studies on deoxy-cyanomet hybrids is withou t foundation.