ISSN:
0887-3585
Keywords:
nickel-iron hybrid hemoglobins
;
two-state allosteric model
;
single crystal absorption spectra
;
Bohr effect
;
multisubunit proteins
;
quaternary structure
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
Oxygen binding by the human hemoglobin tetramer in the T quaternary structure 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; Rivetti et al., Biochemistry 32:2888-2906, 1993). However, cooperativity within the tetramer can be masked by a difference in affinity between the α and β subunits. Indeed, analysis of the binding curves derived from absorption of light polarized along two different crystal directions, for which the projections of the α and β hemes are slightly different, revealed an inequivalence in the intrinsic oxygen affinity of the α and β subunits (p50(α) ≅ 80 torr, p50(β) ≅ 370 torr at 15°C) that compensates a small amount 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oxygen binding curves of single crystals of hemoglobin (in a different lattice) in which the iron in the α subunits has been replaced by the non-oxygen-binding nickel(II). The Hill n is 0.90 ± 0.06, and the p50 is slightly different for light polarized parallel to different crystal directions, indicating a very small difference in affinity between the two crystallographically inequivalent β subunits. The average crystal p50 is 110 ± 20 torr at 15°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 β subunits of the unsubstituted tetramer. These results suggest that Rivetti et al., if anything, overestimated the α/β inequivalence. They therefore did not underestimate the cooperativity within the T quaternary structure, when they concluded that it represents a small deviation from the perfectly noncooperative binding of an MWC allosteric model. Our conclusion of nearly perfect MWC behavior for binding to the T state of unmodified hemoglobin raises the question of the relevance of the large T-state cooperativity inferred for cyanide binding to partially oxidized hemoglobin (Ackers et al., Science 255:54-63, 1992). © 1996 Wiley-Liss, Inc.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.3
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