Structural basis of the lactate-dependent allosteric regulation of oxygen binding in arthropod hemocyanin.
Hemocyanin (Hc) is an oxygen carrier protein in which oxygen binding is regulated by allosteric effectors such as H(+) and L-lactate. Isothermal titration calorimetric measurements showed that L-lactate binds to dodecameric and heterohexameric Hc and to the CaeSS3 homohexamer but not to the CaeSS2 monomer. The binding of lactate caused no change in the optical absorption and x-ray absorption spectra of either oxy- or deoxy-Hc, suggesting that no structural rearrangement of the active site occurred. At pH 6.5, the oxygen binding rate constant k(obs) obtained by flash photolysis showed a significant increase upon addition of L-lactate, whereas L-lactate addition had little effect at pH 8.3. Lactate binding caused a concentration-dependent shift in the interhexameric distances at pH 6.5 based on small angle x-ray scattering measurements. These results show that L-lactate affects oxygen affinity at pH 6.5 by modulating the global structure of Hc without affecting its binuclear copper center (the active site). In contrast to this, the active site structure of deoxy-Hc is affected by changes in pH (Hirota, S., Kawahara, T., Beltramini, M., Di Muro, P., Magliozzo, R. S., Peisach, J., Powers, L. S., Tanaka, N., Nagao, S., and Bubacco, L. (2008) J. Biol. Chem. 283, 31941-31948). Upon addiction of lactate, the kinetic behavior of oxygen rebinding for Hc was heterogeneous under low oxygen concentrations at pH 6.5 due to changes in the T and R state populations, and the equilibrium was found to shift from the T toward the R state with addition of lactate.
Hirota
Shun
S
Graduate School of Materials Science, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Tanaka
Naoki
N
Micetic
Ivan
I
Di Muro
Paolo
P
Nagao
Satoshi
S
Kitagishi
Hiroaki
H
Kano
Koji
K
Magliozzo
Richard S
RS
Peisach
Jack
J
Beltramini
Mariano
M
Bubacco
Luigi
L
D000495:Allosteric Site; D000818:Animals; D001181:Arthropods; D001665:Binding Sites; D001692:Biological Transport; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D006433:Hemocyanins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007773:Lactates / Q000737:chemistry*; D008954:Models, Biological; D010100:Oxygen / Q000737:chemistry*; D011485:Protein Binding; D056928:X-Ray Absorption Spectroscopy / Q000379:methods
D007773:Lactates; D006433:Hemocyanins; D010100:Oxygen
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