Abstract
Recent crystallographic studies suggested that fully liganded human hemoglobin can adopt multiple quaternary conformations that include the two previously solved relaxed conformations, R and R2, whereas fully unliganded deoxyhemoglobin may adopt only one T (tense) quaternary conformation. An important unanswered question is whether R, R2, and other relaxed quaternary conformations represent different physiological states with different oxygen affinities. Here, we answer this question by showing the oxygen equilibrium curves of single crystals of human hemoglobin in the R and R2 state. In this study, we have used a naturally occurring mutant hemoglobin C (β6 Glu→Lys) to stabilize the R and R2 crystals. Additionally, we have refined the x-ray crystal structure of carbonmonoxyhemoglobin C, in the R and R2 state, to 1.4 and 1.8 Å resolution, respectively, to compare precisely the structures of both types of relaxed states. Despite the large quaternary structural difference between the R and R2 state, both crystals exhibit similar noncooperative oxygen equilibrium curves with a very high affinity for oxygen, comparable with the fourth oxygen equilibrium constant (K(4)) of human hemoglobin in solution. One small difference is that the R2 crystals have an oxygen affinity that is 2-3 times higher than that of the R crystals. These results demonstrate that the functional difference between the two typical relaxed quaternary conformations is small and physiologically less important, indicating that these relaxed conformations simply reflect a structural polymorphism of a high affinity relaxed state.
Highlights
This view was largely supported by crystallographic evidence that only one quaternary conformation has so far been observed in a number of different crystals forms of human wild-type, mutant, and chemically modified deoxyhemoglobins [3,4,5,6]
Recent NMR studies using 15N-1H residual dipolar couplings show that the quaternary conformation of human carbonmonoxyhemoglobin in solution is a dynamic ensemble of different quaternary structures that include the crystallographically identified R and R2 states [13]
We show that the use of thin flat crystals of hemoglobin C (6 Glu3 Lys) in the R and R2 state can overcome most of these problems, enabling determination of their oxygen dissociation curves
Summary
Preparation of Hemoglobin C—CS human blood was kindly obtained from Dr Kazuhiko Adachi (The Children’s Hospital of Philadelphia, Philadelphia, PA) and stripped as reported previously. Crystals of carbonmonoxyhemoglobin C in the R2 state were obtained, using the same batch method at 20 °C, from CO-saturated 1.0% (w/v) hemoglobin solutions containing 11% (w/v) PEG 3350, 50 mM HEPES buffer, pH 7.6, and 10% (v/v) glycerol under complete anaerobic conditions. Both types of hemoglobin C crystals grew rapidly to their maximum dimensions within a few day. Among grown crystals of various shapes, thin flat crystals were chosen
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