Abstract
Hemocyanins are multisubunit respiratory proteins found in many invertebrates. They bind oxygen highly cooperatively. However, not much is known about the structural basis of this behavior. We studied the influence of the physiological allosteric effector l-lactate on the oxygenated quaternary structure of the 2x6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS). The presence of 20 mm l-lactate resulted in different scattering curves compared with those obtained in the absence of l-lactate. The distance distribution functions p(r) indicated a more compact molecule in presence of l-lactate, which is also reflected in a reduction of the radius of gyration by about 0.2 nm (3%). Thus, we show for the first time on a structural basis that a hemocyanin in the oxy state can adopt two different conformations. This is as predicted from the analysis of oxygen binding curves according to the "nesting" model. A comparison of the distance distribution functions p(r) obtained from SAXS with those deduced from electron microscopy revealed large differences. The distance between the two hexamers as deduced from electron microscopy has to be shortened by up to 1.1 nm to agree well with the small angle x-ray curves.
Highlights
Hemocyanins are the oxygen transport proteins of most molluscs and arthropods
We studied the influence of the physiological allosteric effector L-lactate on the oxygenated quaternary structure of the 2؋6-meric hemocyanin from the lobster Homarus americanus employing small angle x-ray scattering (SAXS)
The radius of gyration was determined from the distance distribution function p(r) (Fig. 2)
Summary
Hemocyanins are the oxygen transport proteins of most molluscs and arthropods. They are enormous molecular structures, ranging in molecular mass from 4.5 ϫ 105 to more than 107 Da; some contain more than 100 oxygen binding sites [1,2,3]. A suitable method to detect conformational changes of large proteins in solution is small angle x-ray scattering (SAXS). This technique has been applied successfully to monitor changes of the quaternary structure for several cooperative proteins such as hemoglobin and a number of allosteric enzymes (10 –18). We will show that the fully oxygenated 2ϫ6-meric hemocyanin can adopt two clearly distinguishable conformations, which differ in structural details This observation indicates that the allosteric influence of L-lactate on the oxygen binding behavior of this hemocyanin is based on a preferential binding of L-lactate to one of the two possible conformations present in the oxy state
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