OXYGEN-BINDING BY SINGLE-CRYSTALS OF HEMOGLOBIN - THE PROBLEM OF COOPERATIVITY AND INEQUIVALENCE OF ALPHA-SUBUNIT AND BETA-SUBUNIT

Oxygen binding by the human hemoglobin tetramer in the T quaternary st ructure is apparently noncooperative in the crystalline state (Hill n= 1.0), as predicted by the two-state allosteric model of Monod, Wyman, and Changeux (MWC) (Mozzarelli et al., Nature 351:416-419, 1991; Rivet ti et al., Biochemistry 32:2888-2906, 1993). However, cooperativity wi thin the tetramer can be masked by a difference in affinity between th e alpha and beta subunits. Indeed, analysis of the binding curves deri ved from absorption of light polarized along two different crystal dir ections, for which the projections of the alpha and beta hemes are sli ghtly different, revealed an inequivalence in the intrinsic oxygen aff inity of the alpha and beta subunits (p50(alpha) approximate to 80 tor r, p50(beta) = 370 torr at 15 degrees C) that compensates a small amou nt of cooperativity (Rivetti et al., Biochemistry 32:2888-2906, 1993). To further investigate this problem, we have measured oxygen binding curves of single crystals of hemoglobin (in a different lattice) in wh ich the iron in the a subunits has been replaced by the non-oxygen-bin ding nickel(II). The Hill n is 0.90+/-0.06, and the p50 is slightly di fferent for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystal lographically inequivalent beta subunits. The average crystal p50 is 1 10+/-20 torr at 15 degrees C, close to the p50 of 80 torr observed in solution, but about threefold less than the p50 calculated by Rivetti et al. (Biochemistry 32:2888-2906, 1993) for the beta subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the alpha/beta inequivalence. They therefore d id not underestimate the cooperativity within the T quaternary structu re, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our concl usion of nearly perfect MWC behavior for binding to the T state of unm odified hemoglobin raises the question of the relevance of the large T -state cooperativity inferred for cyanide binding to partially oxidize d hemoglobin (Ackers et al., Science 255:54-63, 1992). (C) 1996 Wiley- Liss, Inc.