Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state

Abstract

Single-crystal ultraviolet spectroscopy, X-ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa.Three types of crystals have been investigated. Type-I monoclinic crystals, which have been used for the X-ray structure determination, contain virtually only deoxyhemocyanin. Type-I1 monoclinic crystals, which are less well ordered than the type-I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type-I1 monoclinic form, subject to gradual conversion to methemocyanin.Hemocyanins are the dioxygen-transporting copper-containing proteins occurring freely dissolved in a large number of invertebrate species. They belong to the class of type-I11 copper proteins, containing a dinuclear copper site. The architecture of the molluscan hemocyanins, large cylinders with molecular mass of up to 10 MDa, differs completely from that of the arthropodan hemocyanins, which are hexamers or multihexamers with molecular mass ranging from 450 kDa to a few million daltons, all built from subunits of z 75 kDa [l -81. In spite of these differences in architecture a wealth of spectroscopic evidence [I, 21 and recent amino acid sequence information [3 -101 indicate that at least half of the dinuclear copper site in the two classes of hemocyanins has similar features.So far, the three-dimensional structure of only one hemocyanin has been solved at atomic resolution by X-ray diffraction techniques: the single-hexameric hemocyanin from the spiny lobster Panulirus interruptus, an arthropod [ll, 121. Using this structure, good progress has recently been made with the structural elucidation of subunit I1 from Limulus polyphemus hemocyanin (Magnus and Lattman, personal communication), but no atomic details have been reported as yet. Amino acid sequence information is now available for ten hemocyanin subunits from six different arthropods. Sequence comparisons show that the Panulirus subunit and its hexamer form the building block of all arthropodan hemocyanins [3 -81. For a meaningful analysis and interpretation of the X-ray structure of P. interruptus hemocyanin with respect to its Abbreviations. XAS, X-ray absorption spectroscopy in the fluorescence mode; Me, SO, dimethylsulfoxide. function, it is very desirable to know the oxidation and oxygenation state of the copper atoms in the crystals used for the structure determination.Spectroscopic studies in solutions have shown that deoxyhemocyanin, which is colourless, contains a pair of Cu(1) ions, while in oxyhemocyanin, which is blue, a pair of Cu(I1) ions is observed [I, 21. In addition, a colourless met form exists containing CU(II)~ without bound oxygen [l, 21. Furthermore, semi-met forms with a Cu(I)/C...

Oxygen binding and activation by the complexes of PY2- and TPA-appended diphenylglycoluril receptors with copper and other metals

“…The XANES of 1a also shows a pre-edge transition, due to excitation of the 1s electron to unoccupied higher orbitals; the position and transition probability of this pre-edge transition are determined by the symmetry of the ligand environment, and hence by the coordination number. 37, 38 The pre-edge transition of 1a was found to be surprisingly weak in comparison to that of the Cu(I) complex of 4, studied previously by our group. 12 This weakness is possibly due to deviations from a symmetric tri-coordinate Cu(I) complex; it is however not clear from the XANES what the exact nature of these deviations is.…”

“…For more dilute solutions, longer exposure to oxygen was needed, but as care had to be taken not to heat the sample too much, this time was limited to 10 min. For example, a sample with a concentration of 1a of 1.0 × 10 −4 M showed an apparent extinction coefficient at 364 nm of 12300 M −1 cm −1 , corresponding to 81% oxygenation; when the XANES of this very sample was simulated with varying contributions of deoxygenated and oxygenated spectra, following a procedure reported before to establish the degree of oxygenation of hemocyanin crystals, 38 exactly the same degree of oxygenation as derived from the UV-vis was found. For a sample of 1b prepared in the same way as previously attempted for oxygenated 3, 12 the XANES (not shown) not only deviated from either pure 1a or 1b, but also from any linear combination thereof, presumably because other oxidized dinuclear Cu species with different XANES features, such as Cu(II) 2 (OH) 2 , had been formed; the extent of oxygenation (or oxidation) was therefore more difficult to determine accurately, but is estimated at between 40 and 70%.…”

Molecular Basis of the Bohr Effect in Arthropod Hemocyanin

“…Each copper atom is pentacoordinated in a square pyramidal geometry, where the equatorial plane is now defined by two histidyl imidazole nitrogens and the bound oxygen, while a third histidyl nitrogen is axially coordinated to copper. All three Cu-N bond lengths are 1.9 Å, and the metal-metal separation is 3.0 -3.5 Å (6), a distance supported by EXAFS analysis for the closely related P. interruptus oxyHc (7,8).…”

Crystallographic Evidence That the Dinuclear Copper Center of Tyrosinase Is Flexible during Catalysis

“…Because the crystal structure of tyrosinase has been undetermined until now, the catalytic mechanism at the atomic level has not yet been elucidated. To build structural models of tyrosinase, it would be practical to reference the structural information from the type 3 copper protein members (5)(6)(7)(8)(9)(10)(11). As a common feature in the active site of these structures, each of two closely spaced copper ions is coordinated by three His residues through N⑀ nitrogen atoms.…”