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

Abstract

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.