Serge N. Vinogradov

A phylogenomic profile of globins

BackgroundGlobins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold.ResultsA census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma.ConclusionAlthough Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.

Diversity of Globin Function: Enzymatic, Transport, Storage, and Sensing

The availability of genomic information from the three kingdoms of life has altered substantially our view of the globin superfamily. It is now evident that Hbs,2 defined as hemeproteins comprising five to eight -helices (A–H), with an invariant His at position F8 providing the proximal ligand to the heme iron, occur as three families in two structural classes (1). Within each family, the Hb can be either chimeric or SD. Historically, the first members of the two families that display the canonical 3/3 -helical fold were chimeric: the FHbs in Escherichia coli and yeast discovered in 1990, consisting of an N-terminal Hb coupled to a ferredoxin reductase-like domain, and the GCSs reported in bacteria and Archaea a decade later, comprising anN-terminalHb linked to variable gene regulatory domains. The third family of Hbs discovered concomitantly in algae, ciliates, and bacteria were the 2/2Hbs (“truncated” Hbs), which exhibit a 2/2 -helical fold (see supplemental figure). More recently, SD globins have been discovered in the FHb-like and sensorHb families that we have called SDFgbs and SDSgbs, respectively (2, 3). Fig. 1 shows diagrammatically the three Hb families and summarizes their distribution in bacteria and eukaryotes. A classification of Hbs is presented in the supplemental table. Only bacteria have representatives of all three families in chimeric and SD guise; the Archaea and eukaryotes lack FHbs and GCSs, respectively. On the basis of the higher sequence similarity to bacterial FHbs/SDFgbs than toGCSs and 2/2Hbs and the presence of FHbs/SDFgbs in unicellular eukaryotes, we have proposed that all eukaryoticHbs, including vertebrate / -globins,Mbs, Ngbs, andCygbs and all the invertebrate and plant Hbs, emerged from one or more ancestral bacterial SDFgbs (2). The variety of Hbs in bacteria makes it clear that the familiar O2 transport function of vertebrate Hbs is a relatively recent adaptation and that the early Hb functions must have been enzymatic andO2-sensing. In this review,wewill not discussO2 transport by animal (metazoan) Hbs; instead, we will focus on the reactions and functions of the FHbs/SDFgbs in the first five sections. The functions of the remaining two globin families will be discussed in the last two sections.

An evolutionary tree for invertebrate globin sequences

SummaryA phylogenetic tree was constructed from 245 globin amino acid sequences. Of the six plant globins, five represented the Leguminosae and one the Ulmaceae. Among the invertebrate sequences, 7 represented the phylum Annelida, 13 represented Insecta and Crustacea of the phylum Arthropoda, and 6 represented the phylum Mollusca. Of the vertebrate globins, 4 represented the Agnatha and 209 represented the Gnathostomata. A common alignment was achieved for the 245 sequences using the parsimony principle, and a matrix of minimum mutational distances was constructed. The most parsimonious phylogenetic tree, i.e., the one having the lowest number of nucleotide substitutions that cause amino acid replacements, was obtained employing clustering and branch-swapping algorithms. Based on the available fossil record, the earliest split in the ancestral metazoan lineage was placed at 680 million years before present (Myr BP), the origin of vertebrates was placed at 510 Myr BP, and the separation of the Chondrichthyes and the Osteichthyes was placed at 425 Myr BP. Local “molecular clock” calculations were used to date the branch points on the descending branches of the various lineages within the plant and invertebrate portions of the tree. The tree divided the 245 sequences into five distinct clades that corresponded exactly to the five groups plants, annelids, arthropods, molluscs, and vertebrates. Furthermore, the maximum parsimony tree, in contrast to the unweighted pair group and distance Wagner trees, was consistent with the available fossil record and supported the hypotheses that the primitive hemoglobin of metazoans was monomeric and that the multisubunit extracellular hemoglobins found among the Annelida and the Arthropoda represent independently derived states.

Spectrophotometric study of several sensitive reagents for serum iron

A comparison of three sensitive ligands for iron is described. All show varying degrees of enhancement of values by copper, an element that is the primary interference encountered in serum assays for iron. Two of three tested reagents, 2,4-bis(5,6-diphenyl-1,2,4-triazin-3-yl) pyridine tetrasulfonate and Ferene S are not current clinical chemistry choices whereas ferrozine is widely used. The described study presents spectral characterization of the copper interference with all three ligands. The virtual complete removal of this interference, by the masking actions of either thioglycolic acid, neocuproine or thiourea, is also described . Evidence will be presented that contradicts a previously proposed mechanism for the action of thioglycolic acid which suggests that copper is prevented from dissociating from its protein-binding site by this compound. The preparation of a protein-free filtrate as the matrix in which to generate the color reaction is described, and alternative procedures discussed in brief.

The dissociation of Lumbricus terrestris hemoglobin: a model of its subunit structure.

Abstract The extracellular hemoglobin of the earthworm Lumbricus terrestris has a molecular weight of 3.68 ± 0.17 · 106 and contains 146 ± 7 heme groups. Its dissociation in the presence of denaturants and at alkaline pH was investigated by gel filtration and polyacrylamide gel electrophoresis. The number and size of the constituent polypeptide chains of the resulting subunits obtained by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were correlated with the previously determined number and size of the constituent polypeptide chains of the hemoglobin (Shlom, J. M. and Vinogradov, S. N., (1973) J. Biol. Chem. 248, 7904–7912). The smallest heme-containing subunit isolated by gel filtration at alkaline pH elutes at a volume corresponding to a molecular weight of 24 000–30 000, and corresponds to the smallest subunit observed previously by sodium dodecyl sulfate polyacrylamide gel electrophoresis (subunit I, mol. wt. approx. 13 000). Analytical gel filtration of the subunit in 6 M guanidine hydrochloride gave a molecular weight of 15 900; its iron content was found to be 0.35 ± 0.011% by weight, corresponding to a minimum molecular weight of 16 000 ± 500. The number of copies of subunit I determined by quantitative electrophoresis of the isolated subunit and the hemoglobin, using the dyes Fast Green and Coomassie Blue, was found to be 74 ± 7 per mol of native hemoglobin. This subunit accounts for approx. 25% of the total molecular weight of Lumbricus hemoglobin and contains 50% of the total number of heme groups. A model of the subunit structure of the hemoglobin is proposed which accounts for the known properties of this molecule and the subunits observed upon its dissociation.

Proton magnetic resonance evidence for methionine-iron coordination in mammalian-type ferrocytochrome c

Abstract Proton magnetic resonance spectra of mammalian-type ferrocytochromes c from nine species ranging from yeast to mammals provide evidence for identical methionine-iron coordination in all these protein molecules.

Bacterial and archaeal globins - a revised perspective.

A bioinformatics survey of putative globins in over 2200 bacterial and some 140 archaeal genomes revealed that over half the bacterial and approximately one fifth of archaeal genomes contain genes encoding globins that were classified into three families: the M (myoglobin-like), and S (sensor) families all exhibiting the canonical 3/3 myoglobin fold, and the T family (truncated myoglobin fold). Although the M family comprises 2 subfamilies, flavohemoglobins (FHbs) and single domain globins (SDgbs), the S family encompasses chimeric globin-coupled sensors (GCSs), single domain Pgbs (protoglobins) and SSDgbs (sensor single domain globins). The T family comprises three classes TrHb1s, TrHb2s and TrHb3s, characterized by the abbreviated 2/2 myoglobin fold. The Archaea contain only Pgbs, GCSs and TrHb1s. The smallest globin-bearing genomes are the streamlined genomes (~1.3Mbp) of the SAR11 clade of alphaproteobacteria and the slightly larger (ca. 1.7Mbp) genomes of Aquificae. The smallest genome with members of all three families is the 2.3Mbp genome of the extremophile Methylacidiphilum infernorum (Verrumicrobia). Of the 147 possible combinations of the eight globin subfamilies, only 83 are observed. Although binary combinations are infrequent and ternary combinations are rare, the FHb+TrHb2 combination is the most commonly observed. Of the possible functions of bacterial globins we discuss the two principal ones - nitric oxide detoxification via the NO dioxygenase or denitrosylase activities and the sensing of oxygen concentration in the environmental niche. In only few cases has a physiological role been demonstrated in vivo. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Circular dichroism studies: I. Cytochrome c

Abstract The circular dichroism spectra of the ferri and ferro forms of cytochrome c obtained from horse, beef, chicken, turtle, and Pseudomonas aeruginosa have been measured at pH 7.0 over the wavelength region from 690 to 220 mμ. For purposes of analysis the spectra were arbitrarily divided into five regions: region I, 690-580 mμ; region II, 580-450 mμ (region related to heme coordination); region III, 450-350 mμ (Soret region); region IV, 350-240 mμ (region of protein-heme interactions and Cotton effects due to the aromatic side-chain residues); and region V, below 240 mμ (region of peptide bond absorption). The spectra are complex, exhibiting numerous Cotton effects. The molar ellipticities of all five cytochromes are small and tend to increase in magnitude at shorter wavelengths. The circular dichroism spectra of the mammalian-type cytochrome c of horse, beef, chicken, and turtle are grossly similar to each other. The similarities and differences between the spectra of the mammalian-type cytochromes c were utilized to locate the positions of some of the optically active transitions in regions II, III, and IV. The positions of the two Cotton effects at 280–290 mμ associated with the aromatic side-chain residues of the protein appear to be unaffected by change in oxidation. The spectra of all five ferrocytochromes c are alike in regions II and III. The spectra of the ferri forms of the mammalian-type and Pseudomonas cytochrome c in the Soret region appear to be mirror images of each other. This effect must reflect a difference in the environment of the heme group in the ferricytochromes c of Pseudomonas and mammalian type. Since the effect does not occur in the spectra of the ferrocytochromes c examined, it is likely that the environment of the heme group in the reduced form of cytochrome c remains invariant. An analysis of the relationship of the circular dichroism of all five ferri- and ferrocytochromes c in regions II and III to the structure of the central coordination complex of mammalian-type cytochrome c is presented.

The structure of invertebrate extracellular hemoglobins (erythrocruorins and chlorocruorins)

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. 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. 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. 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. 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%.(ABSTRACT TRUNCATED AT 400 WORDS)

Removal of sodium dodecyl sulfate from proteins

Effective removal of sodium dodecyl sulfate from proteins in water or sodium phosphate buffer was achieved by column chromatography using the ion-retardation resin AG11A8. An average recovery of 83% protein was obtained, while 0.1 to 1.4 moles of sodium dodecyl sulfate remained on each mole of protein.