Crystal structure of deoxygenated limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: Clues for a mechanism for allosteric regulation

Abstract

The crystal structure of Limulus polyphemus subunit type I1 hemocyanin in the deoxygenated state has been determined to a resolution of 2.18 A . Phase information for this first structure of a cheliceratan hemocyanin was obtained by molecular replacement using the crustacean hemocyanin structure of Panulirus interruptus. The most striking observation in the Limulus structure is the unexpectedly large distance of 4.6 A between both copper ions in the oxygen-binding site. Each copper has approximate trigonal planar coordination by three histidine NE atoms. No bridging ligand between the copper ions could be detected. Other important new discoveries are (1) the presence of a cis-peptide bond between Glu 309 and Ser 310, with the carbonyl oxygen of the peptide plane hydrogen bonded to the N6 atom of the copper B ligand His 324; (2) localization of a chloride-binding site in the interface between the first and second domain; (3) localization of a putative calcium-binding site in the third domain. Furthermore, comparison of Limulus versus Panulirus hemocyanin revealed considerable tertiary and quaternary rigid body movements, although the overall folds are similar. Within the subunit, the first domain is rotated by about 7.5" with respect to the other two domains, whereas within the hexamer the major movement is a 3.1 O rotation of the trimers with respect to each other. The rigid body rotation of the first domain suggests a structural mechanism for the allosteric regulation by chloride ions and probably causes the cooperative transition of the hexamer between low and high oxygen affinity states. In this postulated mechanism, the fully conserved Phe 49 is the key residue that couples conformational changes of the dinuclear copper site into movements of the first domain.

Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

“…This allows us to assume that the deoxy sites in the photolysis experiments are sensitive to pH in a similar way in a hexamer that is oxygenated or deoxygenated. Central to this idea is the fact that all oxygenated type III structures reported from x-ray crystal data are superimposable, despite the fact that the Hcs are from organisms that belong to different phyla, and the proteins may have been crystallized at different pH values (3)(4). Oxygen dissociation occurs from this unique structure, which would explain the pH independence of k off values found here.…”

“…Therefore, our flash photolysis approach does not require any correction for pH effects on the spectrum of oxyHc. 4 The absorbance change upon photolysis of oxyHc at pH 7.1 under O 2 concentrations lower than 0.35 mM could not be successfully fit with a single exponential curve (supplemental Fig. S1, depicts the case for [O 2 ] ϭ 0.14 mM), although single exponential fits were adequate for Hc at pH 8.3 under all oxygen concentrations studied, and at pH 7.1 under higher oxygen concentrations ([O 2 ] Ͼ 0.49 mM).…”

“…In further support of this concept, it should be noted that the presence of a Bohr effect for the binding of carbon monoxide to P. interruptus deoxyHc (36), a ligand that binds to only one copper, can be rationalized by a change in coordination geometry of a single Cu(I) with pH. Previous suggestions have focused instead on a change in copper-copper distance to explain the phenomenon (4,6).…”

“…The active site structures of the deoxy form have been described for two arthropod Hcs, Panulirus interruptus (3) and Limulus polyphemus (subunit II) (4). However, a comparison of the results is limited by the different solution conditions from which the proteins were crystallized.…”

Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

“…There is an additional solvent molecule coordinated to both of the copper atoms in PPO2 only, at an average distance of 2.57 Å. The 2 copper atoms in both PPO1 and PPO2 are separated by a large distance (CuA-CuB distances in PPO1 and PPO2 are 4.53 Å and 4.87 Å, respectively), which resembles the deoxy form of other type 3 copper proteins, such as arthropod hemocyanins (38). Therefore, the current PPO structure represents the inactive deoxy state of the enzyme.…”