Using a new template based on an alignment of 145 nonvertebrate globins we examined several recently determined sequences of putative globins and globin-like hemeproteins. We propose that all globins have evolved from a family of ancestral, approx. 17-kDa hemeproteins, which displayed the globin fold and functioned as redox proteins. Once atmospheric O2 became available the acquisition of oxygen-binding properties was initiated, culminating in the various highly specialized functions known as present. During this evolutionary process, we suggest that (1) high oxygen affinity may have been acquired repeatedly and (2) the formation of chimeric proteins containing both a globin and a flavin binding domain was an additional and distinct evolutionary trend. Furthermore, globin-like hemeproteins encompass hemeproteins produced through convergent evolution from nonglobin ancestral proteins to carry out O2-binding functions as well as hemeproteins whose sequences exhibit the loss of some or all of the structural determinants of the globin fold. We also propose that there occurred two cases of horizontal globin gene transfer, one from an ancestor common to the ciliates Paramecium and Tetrahymena and the green alga Chlamydomonas to a cyanobacterium ancestor and the other, from a eukaryote ancestor of the yeasts Saccharomyces and Candida to a bacterial ancestor of the proteobacterial genera Escherichia, Alcaligenes, and Vitreoscilla.

Seven-hundred globin sequences, including 146 nonvertebrate sequences, were aligned on the basis of conservation of secondary structure and the avoidance of gap penalties. Of the 182 positions needed to accommodate all the globin sequences, only 84 are common to all, including the absolutely conserved PheCD1 and HisF8. The mean number of amino acid substitutions per position ranges from 8 to 13 for all globins and 5 to 9 for internal positions. Although the total sequence volumes have a variation -2-3%, the variation in volume per position ranges from -13% for the internal to -21% for the surface positions. Plausible correlations exist between amino acid substitution and the variation in volume per position for the 84 common and the internal but not the surface positions. The amino acid substitution matrix derived from the 84 common positions was used to evaluate sequence similarity within the globins and between the globins and phycocyanins C and colicins A, via calculation of pairwise similarity scores. The scores for globin-globin comparisons over the 84 common positions overlap the globinphycocyanin and globin-colicin scores, with the former being intermediate. For the subset of internal positions, overlap is minimal between the three groups of scores. These results imply a continuum of amino acid sequences able to assume the common three-on-three a-helical structure and suggest that the determinants of the latter include sites other than those inaccessible to solvent.

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The complete dissociation of the hexagonal bilayer structure ofLumbricus terrestris hemoglobin (3900 kDa) at neutral pH, in the presence of urea, guanidine hydrochloride, sodium perchlorate, potassium thiocyanate, sodium phosphotungstate, and sodium phosphomolybdate, followed by gel filtration at neutral pH on Sephacryl S-200 or Superose 6, produced two fragments, II (65 kDa) and m (17 kDa); NaDodSO4/polyacrylamide gel electrophoresis showed that peak II consisted of subunits D1 (31 kDa, chain V), D2 (37 kDa, chain VI), and T (50 kDa, disulfide-bonded trimer of chains H, HI, and IV) and that peak II consisted of subunit M (16 kDa, chain 1). When dissociation was incomplete, two additional peaks were present, peak Ia eluting at the same volume as the whole hemoglobin and peak lb (200 kDa). Scanning transmission electron micrographs of peak Ia showed it to consist of whole molecules and of incomplete hexagonal bilayer structures, missing an apparent 1/12th. Peak lb contained all four subunits but was usually deficient in subunits D1 and D2, was not always in equilibrium with the whole molecule, and could be dissociated further into II and HI. The patterns of dissociation observed at neutral pH were very similar to those observed previously at alkaline pH and at acid pH and appear to be incompatible with the generally accepted multimeric model of Lumrbncus hemoglobin subunit structure. A model is proposed in which it is postulated that the stoichiometries of some of the subunits need not be constant and that subunits D1 and D2 either form a "bracelet" decorated with complexes of T and M subunits or serve as "linkers" between the latter, to provide the appearance of a two-tiered hexagonal structure. Additional support for the proposed model comes from observations that the fragment II obtained subsequent to dissociation at pH 4, in sodium phosphotungstate, in sodium perchlorate, and in potassium thiocyanate was found to be in equilibrium with a hexagonal bilayer structure IaR(II), whose dimensions were =20% smaller than those of the native hemoglobin.

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