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Oxygen binding by α(Fe2+)2β(Ni2+)2 hemoglobin crystals

Published online by Cambridge University Press:  01 April 2000

STEFANO BRUNO
Affiliation:
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy
STEFANO BETTATI
Affiliation:
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy
MICHELE MANFREDINI
Affiliation:
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy
ANDREA MOZZARELLI
Affiliation:
Institute of Biochemical Sciences, University of Parma, 43100 Parma, Italy National Institute for the Physics of Matter, University of Parma, 43100 Parma, Italy
MARTINO BOLOGNESI
Affiliation:
Center of Advanced Biotechnology-IST and Department of Physics, National Institute for the Physics of Matter, University of Genova, 16132 Genova, Italy
DANIELA DERIU
Affiliation:
Center of Advanced Biotechnology-IST and Department of Physics, National Institute for the Physics of Matter, University of Genova, 16132 Genova, Italy
CAMILLO ROSANO
Affiliation:
Center of Advanced Biotechnology-IST and Department of Physics, National Institute for the Physics of Matter, University of Genova, 16132 Genova, Italy
ANTONIO TSUNESHIGE
Affiliation:
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
TAKASHI YONETANI
Affiliation:
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
ERIC R. HENRY
Affiliation:
Laboratory of Chemical Physics, NIDDK, NIH, Bethesda, Maryland 20892
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Abstract

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of α and β subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of α and β hemes separately. Previous studies have characterized the oxygen binding by α(Ni2+)2 β(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of α(Fe2+)2 β(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 ± 3 and 87 ± 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 ± 0.06 and 0.90 ± 0.03. Analysis of the binding curves, taking into account the different projections of the α hemes along the optical directions, indicates that the oxygen affinity of α1 hemes is 1.3-fold lower than α2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the β subunits of α(Ni2+)2 β(Fe2+)2 crystals. The average oxygen affinity of the α subunits (p50 = 91 Torr) is about 1.2-fold higher than the β subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.

Type
Research Article
Copyright
© 2000 The Protein Society

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