Hexagonal Bilayer Structure Research Articles

Significant magnetism enhancement and weak spin-orbit coupling effect in Mn13-nCon (n = 0–13) bimetal alloy clusters

Abstract The ground state structures, spin, orbital magnetic moments and magnetic anisotropies of bimetallic clusters Mn13-nCon (n = 0–13) are systematically investigated by using density functional theory (DFT) with and without spin-orbit coupling. The studies reveal a transition from icosahedron (ICO) to the hexagonal bilayer (HBL) structure, following the rule putting Mn in a central position and Co in the surface of icosahedrons. The magnetic moments of clusters increase to the maximum in Mn9Co4 (39µB) and then decrease as a function of Co concentration. Besides, we find that the orbital moments of all clusters are dominated by Co atoms, as the composition of Co increases, the orbital moments increase from 0.01µB (Mn13) to 1.34µB (Co13). The magnetic anisotropies and magnetic anisotropy energies (MAEs) are tiny in all clusters.

High thermoelectric performance in the hexagonal bilayer structure consisting of light boron and phosphorus elements

Two-dimensional layered materials have attracted tremendous attentions due to their extraordinary physical and chemical properties. Using first-principles calculations and Boltzmann transport theory, we give an accurate prediction of the thermoelectric properties of boron phosphide (BP) bilayer, where the carrier relaxation time is treated within the framework of electron-phonon coupling. It is found that the lattice thermal conductivity of BP bilayer is much lower than that of its monolayer structure, which can be attributed to the presence of van der Waals interactions. On the other hand, the graphene-like BP bilayer shows very high carrier mobility with a moderate band gap of 0.88 eV. As a consequence, a maximum p-type ZT value of ~1.8 can be realized along the x-direction at 1200 K, which is amazingly high for systems consisting of light elements only. Moreover, we obtain almost identical p- and n-type ZT of ~1.6 along the y-direction, which is very desirable for fabrication of thermoelectric modules with comparative efficiencies. Collectively, these findings demonstrate great advantages of the layered structures containing earth-abundant elements for environment-friendly thermoelectric applications.

Spin-orbit coupling effect on structural and magnetic properties of ConRh13−n (n = 0–13) clusters

The effect of spin-orbit interaction on the structures and magnetism of ConRh13−n (n = 0–13) clusters have been systematically investigated by using the spin-orbit coupling (SOC) implementation of the density functional theory (DFT). The results calculated without SOC (NSOC) show that Rh13 prefers the double simple-cubic configuration, and icosahedron is the favorable structure for n = 1–9, while n ≥ 10, clusters favor the hexagonal bilayer structure. The inclusion of SOC in calculation does not change the geometries of clusters. Compared with that in NSOC calculation, although the binding energy per atom in clusters with same composition decreases in SOC calculation, the relative stability of clusters with different compositions does not change. An interesting result is that the spin moments of clusters for n = 1–9 are almost constant (21 μB). Spin-orbit interaction recovers orbital moment and its anisotropy by removing crystal-field effect in calculation. The destruction of bonding symmetry and relaxation of bonding account for high anisotropies of orbital moments in Co11Rh2 and CoRh12 clusters. With atomic composition (Co/Rh) around 4/9–5/8 and 9/4, the Co-Rh clusters exhibit high magnetic anisotropy energies.

Experimental and theoretical studies on compressive deformation characteristics of particle aggregates in water

The compressive deformation characteristics of a single aggregate of polystyrene particles were studied both experimentally and theoretically. Experimental apparatus to measure the mechanical characteristics of a single aggregate with in situ observation of the compression behavior of the aggregate was constructed. Then, the time variations of the compressive force and deformation behavior of the aggregate during simple compression were investigated. In addition, discrete element method (DEM) simulations were carried out to numerically demonstrate the compression behavior of an aggregate between two planes, where the time variation of the compressive force of the aggregate as well as the area strain of each particle in the aggregate can be predicted. The largest spike-like peak of the compressive force appeared at the stage where an aggregate with a hexagonal bilayer structure was formed, as observed experimentally. The compressive force of the aggregate increased as the number of particles in the aggregate increased or the diameter of the primary particles decreased. An increase in the frictional coefficient between primary particles caused an increase in the compressive force of the aggregate for the entire period of compression, but the frictional coefficient between a particle and a wall did not affect the compressive deformation behavior of the aggregate.

The self-association of the giant hemoglobin from the earthworm, Lumbricus terrestris

Background: The crystallographic structure of the gigantic hemoglobin (erythrocruorin) of the annelid worm, Lumbricus terrestris, provides a molar mass of 3.6MDa for the hexagonal bilayer structure. Prior to this determination, some light-scattering and ultracentrifugal measurements indicated higher masses: 4.1–4.4MDa. Values of 3.6MDa were attributed to dissociation or subunit loss. However, early electron microscopy of the giant hemoglobin from a related annelid, Eumenia crassa by Öster Levin, showed that the hexagonal bilayer molecules were present mostly as oligomers; few were monomeric. Methods: Measurements by light-scattering of solutions of Lumbricus hemoglobin resolved by size-exclusion chromatography have been used to determine the weight-average molar mass of self-associating proteins. The X-ray structure has been re-examined. Results: Our measurements show that both 3.6MDa monomers and self-association products are present as a mixture. Analysis of the X-ray structure indicates several different kinds of monomer–monomer interactions. Conclusions: We propose that the measured masses of Lumbricus hemoglobin as high as 4.4MDa, result from oligomerization. These masses would result from the presence of an array of oligomers of various sizes together with monomers of 3.6MDa. Furthermore, several different kinds of monomer–monomer interactions are clearly evident in the X-ray structure as well as in solution. General significance: The results demonstrate that self-association of monomers of the hemoglobin of Lumbricus terrestris explains the high molar masses of 4.1–4.4MDa previously observed.

Influence of water on the properties of an Au/Mpy/Pd metal/molecule/metal junction

The geometric and electronic structure of the metal–molecule interface in metal/molecule/metal junctions is of great interest since it affects the functionality of such units in possible nanoelectronic devices. We have investigated the interaction between water and a palladium monolayer of a Au(111)/4-mercaptopyridine/Pd junction by means of DFT calculations. A relatively strong bond between water and the palladium monolayer of the Au/Mpy/Pd complex is observed via a one-fold bond between the oxygen atom of the water molecule and a Pd atom. An isolated H2O molecule adsorbs preferentially in a flat-lying geometry on top of a palladium atom that is at the same time also bound to the nitrogen atom of a Mpy molecule of the underlying self-assembled monolayer. The electronic structure of these Pd atoms is considerably modified which is reflected in a reduced local density of states at the Fermi energy. At higher coverages, water can be arranged in a hexagonal ice-like bilayer structure in analogy to water on bulk metal surfaces, but with a much stronger binding which is dominated by O–Pd bonds.

Hcp metal nanoclusters with hexagonalA−Abilayer stacking stabilized by enhanced covalent bonding

First-principles total energy calculations within density functional theory have been performed to study the geometric and electronic structures of ${\text{Ru}}_{n}$ nanoclusters of varying size $n$ $(14\ensuremath{\le}n\ensuremath{\le}42)$. Strikingly, for the size range of $n=14$ to 38, the clusters always prefer a hexagonal bilayer structure with $A\text{\ensuremath{-}}A$ stacking, rather than some of the more closely packed forms, or bilayer with $A\text{\ensuremath{-}}B$ stacking. Such an intriguing ``molecular double-wheel'' form is stabilized by substantially enhanced interlayer covalent bonding associated with strong $s\text{\ensuremath{-}}d$ hybridization. Similar $A\text{\ensuremath{-}}A$ stacking is also observed in the ground states or low-lying isomers of the clusters composed of other hcp elements, such as Os, Tc, Re, and Co. Note that these ``molecular double-wheels'' show enhanced chemical activity toward ${\text{H}}_{2}\text{O}$ splitting relative to their bulk counterpart, implying its potential applications as nanocatalysts.

Synthesis and band gap of ZnO particles with hexagonal bilayer structure

The unique water/PVP (polyvinylpyrrolidone)/n-pentanol interface has been developed to prepare the ZnO particles with hexagonal bilayer structure. By modifying the interface through varying the amount of PVP and water, one can readily tune the particle size and change the particle shape from hexagonal bilayer to capped potlike to hemispherical features. The study of the growth dynamics and extinction spectra suggests that the bilayer structure arises from the selective adsorption of PVP on the ZnO crystallographic planes. Both the photoluminescence and extinction spectra show that the band gap of the hexagonal bilayer ZnO particles shrinks with increasing particle size.

The sulfide binding function of annelid hemoglobins: relic of an old biosystem?

Invertebrates living in sulfide-rich environments have developed different strategies of coping with sulfide toxicity. Some bivalves and annelids have hemoglobins that are capable of binding sulfide for detoxification and/or transporting it to internal bacterial symbionts. Annelids living in the sulfide-rich environments have giant (∼3.6 MDa) hemoglobin, consisting of 144 globin chains arranged in a hexagonal bilayer structure held together by 36 nonglobin linker chains. Some globin chains contain either a free cysteine residue at positions Cys + 1 or at position Cys + 11 relative to the E7 distal residue in the E helix and EF interhelical region, respectively, which bind sulfide. The hexagonal bilayer hemoglobins of annelids living in environments lacking sulfide, do not have the corresponding free cysteine residues and cannot bind sulphide. Given that the early stages of life occurred under anoxic conditions in the presence of sulfide, it is possible that the sulfide binding function from modern annelid globins inhabiting sulphide rich habitats is an evolutionary relic. This proposal seems supported by the recent finding of “protoglobins” which also have a corresponding cysteine residue in Archea known to exist in hyperthermophilic and sulfide-rich environments.

Band structure symmetry effects in Gd multilayers grown on W(1 1 2)

We present evidence of coverage-dependent band structure symmetry effects in scanning tunneling microscopy. We find that the symmetry of the Gd induced band located at about 2 eV below the Fermi energy, at the surface Brillouin zone center, appears to be strongly dependent on the atomic structure in the bilayer regime for Gd on W(1 1 2). Light polarization dependent photoemission from this band indicates more d z 2 symmetry character for rectangular Gd structures and rather more d xz d yz character for the quasi hexagonal bilayer structure. Band symmetry changes accompanying the structural phase transition from rectangular to quasi-hexagonal Gd bilayer structures are consistent with arguments used to explain the apparent STM corrugation observed for Gd on W(1 1 2) (Surf Sci 520(2002)43).

Electron induced restructuring of crystalline ice adsorbed on Pt(1 1 1)

Water adsorbs on Pt(1 1 1) at 137 K to form a series of ordered hexagonal bilayer structures. At submonolayer coverages the ice coalesces to form islands with an ( 37 × 37 ) R25.3° structure which are relatively stable to low energy electrons. At saturation the overlayer compresses to form a ( 39 × 39 ) R16.1° overlayer. Further water condenses on this template to grow multilayers which become metastable with respect to an incommensurate bulk ice film. The √39 structure is unstable to electron exposure, a monolayer film restructuring to form islands of incommensurate bulk ice and exposing bare Pt. As the ice film becomes thicker, the lateral stress increases and relaxation becomes fast. Workfunction measurements show no evidence for any preferential alignment of the water dipole in the epitaxial phases, as compared to bulk ice, indicating that the films are proton disordered. Stress in the epitaxial overlayer is due to the lateral compression which maximises the binding of the first layer to Pt, rather than an ordering of the water dipoles.

Protein and Gene Structure of a Chlorocruorin Chain of Eudistylia vancouverii

The polychaete annelid, Eudistylia vancouverii, contains as oxygen carrier a hexagonal bilayer (HBL) chlorocruorin. One of the globin chains, chain a1, has 142 amino acids (Mr 16,054.99) and its sequence deviates strongly from other nonvertebrate globin sequences. Unprecedented, it displays a Phe at the distal position E7 as well as at position B10, creating a very hydrophobic heme pocket probably responsible for the low oxygen affinity of the native molecule. Phylogenetic analysis of annelid globin chains clearly proves that globin chain a1 belongs to type I of globin chains having a pattern of 3 cysteine residues essential for the aggregation into a HBL structure. The gene coding for globin chain a1 is interrupted by 2 introns at the conserved positions B12.2 and G7.0. Based on protein and gene structure it can therefore be concluded that the globin chains of chlorocruorins are not fundamentally different from other annelid globin chains.

Occurrence of two architectural types of hexagonal bilayer hemoglobin in annelids: comparison of 3D reconstruction volumes of Arenicola marina and Lumbricus terrestris hemoglobins

A 3D reconstruction at 25 Å resolution of native hemoglobin of the polychaete worm Arenicola marina was carried out from frozen-hydrated specimens examined in the electron microscope. The reconstruction volume of this large extracellular multimeric respiratory pigment appears as a hexagonal bilayer structure with eclipsed vertices in its upper and lower hexagonal layers. Conversely, in hemoglobins of oligochaetes, achaetes, and vestimentiferans and in chlorocruorins of the Sabellidae (polychaete) family, the vertices of the upper layer are 16° clockwise rotated with respect to those of the lower layer. The fact that two other polychaete hemoglobins ( Alvinella pompejana and Tylorrhynchus heterochaetus) have the same architecture as Arenicola led us to define two types of hexagonal bilayer hemoglobins/chlorocruorins: (i) type-I present in oligochaete, achaete, and vestimentiferan hemoglobins and in Sabellidae chlorocruorins; and (ii) type-II present in polychaete hemoglobins. A comparative study of the hemoglobins of Lumbricus terrestris (type-I) and Arenicola marina (type-II) showed that only two small differences located in the c4 and c5 linking units are responsible of the important architectural difference present in oligomers. A likely scheme proposed to explain the phylogenic distribution of the two types suggests that Clitellata, Sabellida (polychaete), and vestimentiferan hemoglobins and chlorocruorins derive from a type-I ancestral molecule, while Terebellida ( Alvinella), Phyllodocida ( Tylorrhynchus), and Scolecida ( Arenicola) and possibly other polychaetes derive from an ancestor molecule with type-II hemoglobin. The architectures of the hollow globular substructures are highly similar in Arenicola and Lumbricus hemoglobins, with 12 globin chains and three linking units (c3a, c3b, and c4). The central piece of Arenicola hemoglobin is an ellipsoid while that of Lumbricus is a toroid. No phylogenic correlation could be found between the structure of the central pieces and the architecture type.

The role of linkers in the reassembly of the 3.6 MDa hexagonal bilayer hemoglobin from lumbricus terrestris

The extent and kinetics of reassembly of the four groups of linkers L1-L4 with 213 kDa subassemblies of twelve globin chains D, (bac)3(d)3, isolated from the ∼3.6 MDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris, was investigated using gel filtration. The reassembled HBL’s were characterized by scanning transmission electron microscopic (STEM) mass mapping and their subunit content determined by reversed-phase chromatography. In reassembly by method (A), the linkers isolated by RP-HPLC at pH ∼2.2 were added to D at neutral pH; in method (B), the linkers were renatured at neutral pH and then added to D. With method (A) the percentage of HBL reassembly varied from ⩾13 % in the absence of Ca(II) to ⩽75 % in 1-10 mM Ca(II). Reassembly to HBL structures whose linker contents, STEM images and masses were similar to the native Hb was observed with all the linkers (⩾75 %), with ternary and binary linker combinations (40-50 %) and with individual linkers producing yields increasing in the following order: L1=1-3 %, L2≈L3=10-20 % and L4=35-55 %. The yield was two- to eightfold lower with method (B), except in the case of linkers L1-L3. Although the reassembly kinetics were always biphasic, with t1/2=0.3-3.3 hours and 10-480 hours, the ratio of the amplitudes fast:slow was 1:0.6 with method (A) and 1:2.5 with method (B). These results are consistent with a scheme in which the slow HBL reassembly is dependent on a slow conversion of linker conformation at neutral pH from a reassembly incompetent to a reassembly competent conformation. Although all the linkers self-associate extensively at neutral pH, forming complexes ranging from dimers to >18-mers, the size of the complex does not affect the extent or rate of reassembly. The oxygen binding affinity of reassembled HBLs was similar to that of the native Hb, but their cooperativity was lower. A model of HBL reassembly was proposed which postulates that binding of linker dimers to two of the three T subunits of D causes conformational alterations resulting in the formation of complementary binding sites which permit lateral self-association of D subassemblies, and thus dictate the formation of a hexagonal structure due to the 3-fold symmetry of D.

A hierarchy of disulfide-bonded subunits: the quaternary structure of Eudistylia chlorocruorin.

The quaternary structure of the cysteine-rich, approximately 3500-kDa chlorocruorin (Chl) from the marine polychaete Eudistylia vancouverii was investigated using maximum entropy deconvolution of the electrospray ionization mass spectra (ESIMS). The native Chl provided two groups of peaks, at approximately 25 and approximately 33 kDa, and one peak at approximately 66 kDa. ESIMS of the reduced and reduced and carbamidomethylated Chl and of its subunits obtained by HPLC provided the complete subunit composition of the Chl. Two groups of nonglobin linker chains were observed: L1a-f (25 000.4, 25 017.9, 25 039.6, 25 057.0, 25 074.4 and 25 096.8 Da) and L2a-d (25 402.7, 25 446.0, 25 461.6 and 25 478.3Da) (+/-2.5 Da), with relative intensities L1:L2 = 5:2. Six globin chains were found, a1, a2, and b1-4, with reduced masses of 16 051.5, 16 172.4, 16 853.5, 17 088.9, 17 161.2 and 17 103.6 (+/-1.0 Da) and relative intensities of 8:4:1:4:2:1, respectively. Disulfide-bonded dimers and a tetramer of globin chains were identified: D1 = a1 + b3 at 33 207.1; D2 at 33 374.1, which had a cysteinylated Cys (a2 + b2 + Cys); and D3 = a1 + b4 at 33 149.4 Da (+/-3.0 Da), with relative intensities D1:D2:D3 = 5:4:1 and T = a1 + a2 + b1 + b2 at 66 154.8 +/- 4.0 Da. A 206-kDa dodecamer subunit obtained by dissociation of the Chl in 4 M urea [Qabar, A. N., et al. (1991) J. Mol. Biol. 222, 1109-1129], was found to consist only of tetramers T. A model was proposed for the Chl, based on a dimer:tetramer ratio of 2:1: four 206-kDa dodecamers (trimer of tetramers) and 48 dimers tethered to a framework of 30 L1 and 12 L2 linker chains. The 144 globin chains (2480 kDa) and 42 linker chains (1059 kDa) provide a total mass of 3539 kDa, in good agreement with the 3480 +/- 225 kDa determined previously by STEM mass mapping. The hierarchy of disulfide-bonded globin subunits observed for Eudistylia Chl provides a built-in heterogeneity of hexagonal bilayer structures.

The Role of the Dodecamer Subunit in the Dissociation and Reassembly of the Hexagonal Bilayer Structure of Lumbricus terrestris Hemoglobin

The dissociation of the approximately 3500-kDa hexagonal bilayer (HBL) hemoglobin (Hb) of Lumbricus terrestris upon exposure to Gdm salts, urea and the heteropolytungstates [SiW11O39]8- (SiW), [NaSb9W21O86]18- (SbW) and [BaAs4W40O140]27- (AsW) at neutral pH was followed by gel filtration, SDS-polyacrylamide gel electrophoresis, and scanning transmission electron microscopy. Elution curves were fitted to sums of exponentially modified gaussians to represent the peaks due to undissociated oxyHb, D (approximately 200 kDa), T+L (approximately 50 kDa), and M (approximately 25 kDa) (T = disulfide-bonded trimer of chains a c, M = chain d, and L = linker chains). OxyHb dissociation decreased in the order Gdm*SCN > Gdm.Cl > urea > Gdm.OAc and AsW > SbW > SiW. Scanning transmission electron microscopy mass mapping of D showed approximately 10-nm particles with masses of approximately 200 kDa, suggesting them to be dodecamers (a+b+c)3d3. OxyHb dissociations in urea and Gdm.Cl and at alkaline pH could be fitted only as sums of 3 exponentials. The time course of D was bell-shaped, indicating it was an intermediate. Dissociations in SiW and upon conversion to metHb showed only two phases. The kinetic heterogeneity may be due to oxyHb structural heterogeneity. Formation of D was spontaneous during HBL reassembly, which was minimal (</= 10%) without Group IIA cations. During reassembly, maximal (approximately 60%) at 10 mM cation, D occurs at constant levels (approximately 15%), implying the dodecamer to be an intermediate.

Molecular Symmetry of the Dodecamer Subunit of Lumbricus terrestrishemoglobin

The principal functional subunit of the ∼3500 kDa extracellular Lumbricus terrestrishemoglobin is a 213 kDa dodecamer of four chemically distinct globin chains, consisting of a non-covalent complex of three trimer submits (disulfide-bonded chains a, band c) and three monomer subunits (chain d). X-ray diffraction of crystals of the dodecamer grown at neutral pH, were found to be monoclinic, with the unit cell dimensions: a=112.3 Å, b=190.0 Å, c=69.6 Å, β=102.0° with h+ k+ l=2 n+ 1 absent, character istic of space group I121. In addition, these crystals exhibit a pseudo trigonal P321 symmetry with unit cell dimensions a=190.5 Å, b=190.5 Å, c=69.5 Å, γ=120.0°. Assuming that the assymetric unit contains an entire dodecamer, a model of the latter was constructed that satisfies the symmetry of the trigonal pseudo cell and is consistent with the symmetry of the I121 crystallographic cell. The resulting model has strong implications concerning the hexagonal bilayer structure of the native hemoglobin.

Mass Spectrometic Composition, Molecular Mass and Oxygen Binding of Macrobdella decoraHemoglobin and its Tetramer and Monomer Subunits

The hexagonal bilayer hemoglobin (Hb) of the leech Macrobdella decorahas a equilibrium sedimentation mass of 3544(±80) kDa. Maximum entropy analysis of the electrospray ionization mass spectra of the Hb show three groups of peaks: two peaks of equal intensity at ∼17 kDa, A (16,770.1 Da) and B (16,841.9 Da); three peaks at ∼24 kDa, C (24,340.1 Da), D (24,398.6 Da) and E (24,420.0 Da) with relative intensities of 1:6:3:, respectively; and three peaks of equal intensities at ∼33 kDa, F (32,586.0 Da), G (32,714.5 Da) and H (32,849.9 Da). Although reduction with dithiothreitol does not affect the masses of peaks A through E, the ∼33 kDa peaks give rise to four new peaks at ∼16 kDa, P (16,052.2 Da), Q (16,537.3 Da), R (16,666.7 Da) and S (16,792.9 Da), indicating that F, G and H represent disulfide-bonded dimers of globin chains, P + Q, P + R and P + S, respectively. The relative intensities of the three groups of peaks are (A + B) to (C + D + E) to (F + G + H) = 0.39:0.26:0.32, and the globin to linker ratio 0.71:0.29 is in good agreement with the ratio 0.72:0.28 obtained by HPLC. The largest functional subunit obtained by dissociation at pH 7 in 4 M urea, is a subunit lacking linker chains with apparent mass 63(±3) kDa. The equilibrium sedimentation profile of this subunit is fitted best as a monomer-dimer-tetramer equilibrium, with association constants K 1,2= 365 l g −1and K 1,4= 8.1 × 10 5l 3g −3. A model of the Hb consisting of a hexagonal bilayer of 36 tetramer and 42 linker subunits provides a total mass and globin to linker ratio closest to the experimental values. Equilibrium O 2binding measurements of the native Hb and its tetramer and monomer subunits were carried out over the pH range 6.6 to 8.0 at 10 and 25 °C, and in the absence and presence of Na +, Mg 2+and Ca 2+. The Hb exhibits a moderately high O 2affinity, P 50= 4.4 torr at pH 7.5 and 25 °C, a high cooperativiity ( n 50∼ 3) and a substantial Bohr effect, φ = Δlog P 50/ΔpH = −0.38. The tetramer subunit has a higher affinity, lower cooperativity and smaller Bohr effect, 1.9 torr, 1.3 to 1.5 and −0.30, respectively. The monomer subunit has a much higher affinity ( P 50= 0.29 torr) and no cooperativity or Bohr effect. Analysis of the O 2binding curves in terms of the two-state (MWC) allosteric model showed a markedly increased K Tin the tetramers compared with the native Hb, resulting in opposite relative Δ Gvalues. Increases in cation (Ca 2+, Mg 2+) concentrations and pH increase O 2binding affinity as well as K Rand K Tof the Hb but have no effect of the properties of either the tetramer or monomer subunits. The results suggest that the overall cooperativity of O 2binding of the Hb consists of the cooperativity of the tetramer subunit and an additional contribution due to the hexagonal bilayer structure, requiring globin and linker chains.

Hierarchy of globin complexes: The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27·5 nm and height of 18·5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass ( M m) of 3480 ± 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0·251 ± 0·021 wt %, corresponding to a minimum M m of 22·4 kDa. Mass spectra and 1nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of ~30 kDa and five chains of ~16 kDa; the two types of subunits occur in the ratio 0·26:0·74(± 0·08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the ~16 kDa chains and fraction D consisted of the ~30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided M m of 208 ± 23 kDa and 65 ± 12 kDa, respectively, in agreement with 191 ± 13 kDa and 67 ± 5 kDa obtained by gel filtration. Particles with M m = 221 + 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the ~30 kDa linker subunits that have 0·26 to 0·47 heme groups/chain, comprises ~65 kDa tetramers and ~200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 ± 4 and 43 ± 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 ± 14 Ca 2+ /mol protein, and was decreased to ~35 % by the presence of 10 to 25 m m-Ca 2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2·5 to 5 m m-Ca 2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca 2+ exhibited very limited (<10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit. Although the circular dichroism (200 to 250 nm) of the chlorocruorin and the dodecamer subunit was similar, with α-helical contents of ~50%, the [ θ] 222 of the tetramer and linker subunits were lower. Our results are compatible with a view of the quaternary structure of Eudistylia chlorocruorin as a hierarchy of globin complexes, wherein 66 to 75 % of the whole molecule is composed of 12 ~ 200 kDa dodecamers, each consisting of a trimer of tetramers of globin chains having an incomplete “myoglobin fold”. The dodecamers are linked into a hexagonal bilayer structure by 30 to 40 Ca 2+ and 30 to 40 heme-deficient, chimeric globin chains.

A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris

Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.