The structure of invertebrate extracellular hemoglobins (erythrocruorins and chlorocruorins)

Abstract

1. 1. The knowledge accumulated over the last 30 years concerning the subunit structures of the invertebrate extracellular hemoglobins permits us to classify them into four distinct groups. 1.1. (A) Single-domain, single-subunit hemoglobins consisting of single, heme-binding polypeptide chains which have a molecular mass of ca. 16 KDa. These molecules are found in multicellular parasitic organisms such as the trematodes Dicrocoelium and Fasciolopsis and in a few insects, namely in the adult Anisops and in the larvae of Chironomus and of Buenoa. 1.2. (B) Two-domain, multi-subunit hemoglobins consisting of 30–37 KDa polypeptide chains each containing two, linearly connected heme-binding domains, which form polymeric aggregates with molecular masses ranging from 250 to 800 KDa. These hemoglobins are found extensively among the carapaced branchiopod crustaceans: Caenestheria, Daphnia and Lepidurus hemoglobins have been found to consist of 10, 16 and 24 two-domain chains, respectively. Judging from their electron microscopic appearances, some of the hemoglobins may possess different molecular symmetries. 1.3. (C) Multi-domain, multi-subunit hemoglobins consisting of two or more polypeptide chains, each comprising many heme-binding domains of ca. 15–20 KDa each. Examples of this class are found among the carapaceless branchiopod crustaceans, the planorbid snails and the clams from the families Astartidae and Carditidae. Artemia hemoglobin consists of two chains of ca. 125 KDa, each containing 8 heme-binding domains. Planorbis and Helisoma hemoglobins possess a molecular mass of ca. 1700 KDa and consist of 10 chains of 170–200 KDa. Astarte and Cardita hemoglobins appear in electron micrographs as rod-like polymers of variable dimensions, 20–30 nm in diameter and 20–100 nm in length and consist of polypeptide chains of ca. 300 KDa. The crustacean and gastropod hemoglobins vary in their electron microscopic appearance and may possess different molecular symmetries. 1.4. (D) Single-domain, multi-subunit hemoglobins consisting of aggregates of several small subunits, some of which are disulfide-bonded and not all of which contain heme. These molecules are widely distributed among the annelids and possibly also among the pogonophores. They are characterized by a two-tiered, hexagonal electron microscopic appearance, with a vertex-to-vertex diameter of 30 nm and a height of 20 nm, an acidic isoelectric point, a sedimentation coefficient of 50–60 S and a low iron content of 0.24 ± 0.03%. This group should also include the chlorocruorins, which, although possessing a slightly altered heme group, appear to be very similar in their appearance and properties. 1.5. 2. Electrophoretic studies of annelid hemoglobins and chlorocruorins suggest that they display different patterns of quaternary structure. Most oligochaete and polychaete hemoglobins consist of monomeric, 16 KDa heme-binding subunits, dimeric, 33–37 KDa subunits, some of which do not bind heme and trimeric, heme-containing ca. 50 KDa subunits which are disulfide-bonded trimers of ca. 17 KDa polypeptide chains. The achaete hemoglobins consist of monomeric, 16 KDa heme-binding subunits and dimeric 30–37 KDa subunits, some of which may not bind heme and some of which may be disulfide-bonded dimers of ca. 17 KDa subunits. The chlorocruorins exhibit two separate patterns of subunits: monomers and disulfide-bonded dimers and tetramers on one hand, and on the other, disulfide-bonded tetramers with reduction-resistant dimers. 1.6. 3. Small hemoglobins which cannot be considered to be extracellular and myoglobins are found in several invertebrate groups as well as in fungi and plants. Monomeric hemoglobin occurs in ciliated protozoa such as Paramecium. The radular muscle myoglobin in most gastropod molluscs is monomeric, except in the neogastropods such as Buccinum where it is dimeric. A hemoglobin-like heme protein is found in fungi such as Candida. Although the hemoglobins found in the root nodules of legumes are monomeric, a dimeric molecule occurs in the root nodule of the non-leguminous plant Parasponia. 1.7. 4. Comparison of the amino acid sequences of globin chains from two different subunits of Lumbricus and Tylorrhynchus hemoglobins, of the monomeric, intracellular hemoglobin of Glycera, of Chironomus component III, of a leghemoglobin from Pisum and of human hemoglobin, showed that the extracellular annelid sequences have 42 amino acid identities with each other, only 15–18 with the Chironomus sequence and 20–25 with the other globin sequences. It is also known that the three-dimensional structures of Glycera and Chironomus hemoglobins and of Lupinus leghemoglobin are very similar to each other and to those of vertebrate globins. In conclusion, it is pointed out that the two main structural themes found among the extracellular invertebrate hemoglobins, namely, aggregation of single-domain subunits on one hand, and on the other, the aggregation of multi-domain subunits, are also found among the hemocyanins, the former theme occurring in arthropod hemocyanins and the latter one in molluscan hemocyanins.