ALLOSTERIC TRANSITION INTERMEDIATES MODELED BY CROSS-LINKED HEMOGLOBINS

THE structural end-points of haemoglobin's transition from its low-oxy gen-affinity (T) to high-oxygen-affinity CR) state, have been well est ablished by X-ray crystallography(1-7), but short-lived intermediates have proved less amenable to X-ray studies, Here we use chemical cross linking to fix these intermediates for structural characterization. We describe the X-ray structures of three haemoglobins, alpha(2) beta(1) S(82)beta, alpha(2) beta(1)Tm(82)beta and alpha(2) beta(1,82)Tm(82)bet a, which were crosslinked between the amino groups of residues beta Va l1 and beta Lys82 by 3,3'-stilbenedicarboxylic acid (S) or trimesic ac id (Tm) while in the deoxy state, and saturated with carbon monoxide b efore crystallization. alpha(2) beta(1)S(82)beta, which has almost nor mal oxygen affinity, is completely in the R-state conformation; howeve r, alpha(2) beta(1)Tm(82)beta and alpha(2) beta(1,82)Tm(82)beta, both of which have low oxygen affinity, have been prevented from completing their transition into the R state and display many features of a tran sitional intermediate, These haemoglobins therefore represent a snapsh ot of the nascent R state.