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GLOBAL JOURNAL OF BIOCHEMISTRY

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Global Journal of Biochemistry | Volume 1 | Issue 1 | December 2010 www.simplex-academic-publishers.com

© 2010 Simplex Academic Publishers. All rights reserved.

Identification and in silico characterization of a putative ancestor to land plant non-symbiotic hemoglobins from the prasinophyceae algae

Micromonas and Ostreococcus

Iván Fernándeza, Serge N. Vinogradovb, Raúl Arredondo-Petera,*

a Laboratorio de Biofísica y Biología Molecular, Departamento de Bioquímica y Biología Molecular,

Facultad de Ciencias, Universidad Autónoma del Estado de Morelos, Ave. Universidad 1001, Col. Chamilpa, 62210 Cuernavaca, Morelos, México

b Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit MI 48201, USA

*Author for correspondence: Raúl Arredondo-Peter, email: [email protected]

Received 20 Apr 2010; Accepted 17 May 2010; Available Online 10 Nov 2010

Abstract

Nucleotide and protein sequences exhibiting high similarity to land plant non-symbiotic hemoglobins (nsHbs) were identified in the genome of the prasinophyceae algae Micromonas pusilla, M. sp. RCC299, and Ostreococcus sp. RCC809. Sequence alignment showed that Micromonas and Ostreococcus nsHb-like globins (Micropusil, Microsp, and Ostreosp nsHbs, respectively) contain the highly conserved Phe B10, Phe CD1, and proximal and distal His in identical positions as in land plant nsHbs. Also, phenetic analysis showed that Micromonas and Ostreococcus nsHb-like globins are closely related to bacterial Hbs and closer to bryophyte than to other land plant nsHbs. This observation suggests that Micromonas and Ostreococcus nsHb-like globins evolved from a bacterial Hb and are ancestral to land plant nsHbs. Modeling of the tertiary structure of Micromonas and Ostreococcus nsHb-like globins showed that these proteins could fold into the canonical 3-on-3 myoglobin-fold. Also, analysis of the distance of distal His to the heme-Fe suggests that predicted Micropusil and Ostreosp nsHbs might be pentacoordinate and that the Microsp nsHb might be hexacoordinate. Keywords: Evolution; Hemoglobin; Land plants; Micromonas; Non-symbiotic; Ostreococcus Abbreviations

fHb, flavohemoglobin; Hb, hemoglobin; Lb, leghemoglobin; Mb, myoglobin; MYA, million of years ago; Ngb, neuroglobin; nsHb, non-symbiotic hemoglobin; tHb, truncated hemoglobin 1. Introduction

Hemoglobins (Hbs) are O2-binding proteins widely distributed in land plants. These proteins have been detected in primitive bryophytes and evolved monocots and dicots [1]. Based on sequence similarity, synthesis in plant organs, and postulated function plant Hbs are classified into three types: symbiotic Hbs, non-symbiotic Hbs (nsHbs), and truncated Hbs (tHbs). Symbiotic Hbs (or leghemoglobins (Lbs) when isolated from legume plants) are specifically synthesized in nodules of N2-fixing plants, and their apparent function is to facilitate the diffusion of O2 to the respiring bacteroids [2, 3]. Non-symbiotic Hbs exist in primitive and evolved plants, are synthesized in diverse organs from normal and stressed plants [4-7], and their apparent function is to modulate redox potentials and levels of NO [8-12]. Sequences coding for tHbs were detected in genomes from primitive

and evolved plants [13] (Vinogradov et al., submitted). In higher plants the thb genes express in vegetative and embryonic organs and in organs from stressed plants [13-15]. The function of tHbs is not yet known, however it was proposed that plant tHbs function similarly to bacterial tHbs by participating in the metabolism of NO [1].

The major events that occurred during the evolution of land plant Hbs were elucidated over the last few years. For example, phylogenetic analysis revealed that nsHbs and Lbs and tHbs evolved through different lineages and that Lbs originated from nshb genes [1, 16-18]. Also, the analysis of moss nsHbs revealed that primitive land plant nshbs are interrupted by 3 introns in the identical position as in all known land plant nshbs, thus showing that the ancestral land plant nshb was interrupted by 3 introns [7, 19]. The structural analysis of primitive nsHbs and Lbs revealed that changes during the evolution of plant non-symbiotic to symbiotic

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Hbs were a hexacoordinate to pentacoordinate transition at the heme prosthetic group, a length decrease at the CD-loop and N- and C-termini regions, and a compaction of the protein into a globular structure [7, 17, 20].

Although the major events during the evolution of land plant nsHbs are mostly known (above), the ancestor of land plant nsHbs has not yet been identified. This information is essential to fully understand the origin and evolution of plant nsHbs. This work reports the identification and characterization of sequences exhibiting high similarity to land plant nsHbs from the genome of the prasinophyceae algae Micromonas pusilla, M. sp. RCC299, and Ostreococcus sp. RCC809. Results suggest that Micromonas and Ostreococcus nsHb-like globins are ancestral to land plant nsHbs, and thus indicate that land plant nsHbs evolved from algal Hbs. 2. Experimental 2.1. Database search

Putative globin sequences and globin domains were identified in the genome of the prasinophyceae algae Micromonas pusilla, M. sp. RCC299, and Ostreococcus sp. RCC809 using keywords and query sequences from moss (Physcomitrella patens and Ceratodon purpureus, Genbank accession numbers AF218049 and AF309562, respectively) nsHbs and the SUPERFAMILY database (http://supfam.mrc-lmb.cam.ac.uk, last accessed on December 2009) [21]. Resulting sequences were subjected to a FUGUE analysis (http://www-cryst.bioc.cam.ac.uk, last accessed on December 2009) [22] to determine the most similar globin structure and presence of proximal His at the myoglobin (Mb)-fold position F8. Putative globins had to satisfy the following criteria: length higher than 100 amino acids, a FUGUE Z score higher than 6 (which corresponds to 99% specificity [22]) with known globin structures, and the presence of proximal His at position F8. Sequences for the Micromonas and Ostreococcus nshb-like globin genes were identified at the JGI (DOE Joint Genome Institute) genome browser (http://www.jgi.doe.gov/, last accessed on December 2009) using scaffold numbers from the SUPERFAMILY database. 2.2. Sequence alignment and phenetic analysis

Fast multiple sequence alignment and cluster analysis of the Micromonas and Ostreococcus nsHb-like globins and selected land plant nsHbs and bacterial Hbs were performed using the Neighbor-Joining method [23] of the Clustal X program [24]. Sequence alignment was manually verified using the

procedure described by Kapp et al. [25] based on the Mb-fold [26]. Sequence similarity and identity values between the Micromonas and Ostreococcus nsHb-like globins and individual Hbs were obtained from pairwise sequence alignments using the BLASTp program [27] from the Genbank database (http://www.ncbi.nlm.nih.gov, last accessed on December 2009). 2.3. In silico analysis of protein sequences

Hydropathy profiles for the Micromonas and Ostreococcus nsHb-like globins and rice Hb1 (Genbank accession number U76030) were obtained using the Kyte-Doolittle method [28] from the ExPaSy web site (http://expasy.ch/tools/, last accessed on December 2009). Other in silico analysis were performed using tools from the ExPaSy web site and consisted of predicting for chloroplast, mitochondrial, and signal peptide targeting sequences (ChloroP 1.1, MitoProt II and SignalP-NN/HMM tools, respectively) and for molecular weight calculation (SOPMA tool) [29]. 2.4. Molecular modeling and analysis of predicted tertiary structures

The tertiary structure of the Micromonas and Ostreococcus nsHb-like globins was modeled using the automated mode of Swiss Model (http://swissmodel.expasy.org/, last accessed on December 2009) and the SwissPDBViewer essentially as described by Gopalasubramaniam et al. [30]. Model reliability was evaluated using the Verify3D [31] option from the Swiss Model workspace. Models were edited using the VMD program [32] and Adobe Photoshop® software. Distance of amino acid residues at the heme prosthetic group were calculated using the distance tool of SwissPDBViewer. 3. Results and Discussion

Recent evidence indicated that land

plant nsHbs originated and evolved from bryophyte-like organisms about 450 MYA [7, 19]. However, the ancestor to land plant nsHbs has not yet been identified. It is accepted that land plants are mostly monophyletic and originated from green algae [33-37]. The availability of genomes sequenced from diverse green algae is a valuable source of information to search for the globin ancestor of land plant nsHbs. 3.1. Detection of prasinophyceae nsHb-like globin sequences in databases

A search for homologs to nsHbs in the algal genomes using the SUPERFAMILY



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database revealed that 3, 4, and 1 sequences similar to land plant nsHbs exist in the genome of the prasinophyceae algae Micromonas pusilla, M. sp. RCC299, and Ostreococcus sp. RCC809, respectively. No nsHb-like sequences were identified in the genome of other green algae, but from the diatom Thalassiosira pseudonana (Table 1). After sequence evaluation only the following three entries satisfied the criteria to be considered authentic globins (i.e. length higher than 100 amino acids, a FUGUE Z score higher than 6 with known globin structures, and the presence of proximal His at position F8): Micromonas pusilla 8393 scaffold 1:397448-397834 (from the JGI Micromonas pusilla CCMP1545 v2.0 genome browser), Micromonas sp. RCC299 84803 scaffold Chr_09:460488-460937 (from the JGI Micromonas sp. RCC299 v2.0 genome browser), and Ostreococcus sp. RCC809 scaffold 15:108499-108972 (from the JGI Ostreococcus RCC809 genome browser). Also, sequence alignment showed that these entries exhibit ~55% similarity to land plant nsHbs (below). Thus, Micromonas pusilla 8393 scaffold 1:397448-397834, Micromonas sp. RCC299 84803 scaffold Chr_09:460488-460937, and Ostreococcus sp. RCC809 scaffold 15:108499-108972 corresponded to nsHb-like globins and were named as Micropusil nsHb, Microsp nsHb, and Ostreosp nsHb, respectively. These entries were selected for in silico analysis.

Analysis of highest sequence similarity and identity by BLASTp in the Genbank database showed that Micropusil, Microsp, and Ostreosp nsHbs are >30 and 50% identical and similar, respectively, to bacterial Hbs and land plant nsHbs (Table 1). These observations indicate that Micropusil, Microsp, and Ostreosp nsHbs are intermediate between bacterial Hbs and land plant nsHbs, and suggest that they evolved from a bacterial Hb and are ancestral to land plant nsHbs. Also, Ostreosp nsHb is >30 and 50% identical and similar, respectively, to animal neuroglobins (Ngbs). This observation suggests that plant nsHbs and Ngbs evolved from a common ancestor prior to the plant-animal divergence, about 1 200 MYA. 3.2. Sequence alignment and phenetic analysis of prasinophyceae, land plant, and bacterial Hbs

Except for Micropusil nsHb (129 amino acids), Microsp and Ostreosp nsHbs (149 and 157 amino acids, respectively) are similar in length to the land plant nsHbs. Sequence alignment of Micropusil, Microsp, and Ostreosp nsHbs with selected bacterial Hbs and land plant nsHbs and Lbs showed that Micromonas and Ostreococcus nsHb-like globins contain the highly conserved Phe B10, Phe CD1, and

proximal and distal His in identical positions as in land plant nsHbs (Figure 1). However, postulated amino acids for the dimer interface in the rice Hb1 structure [38] are not conserved in Micropusil, Microsp, and Ostreosp nsHbs, which suggests that Micromonas and Ostreococcus nsHb-like globins are monomers in vivo.

Phenetic analysis showed that Micropusil, Microsp, and Ostreosp nsHbs cluster with bacterial Hbs (Figure. 2). This observation shows that Micromonas and Ostreococcus nsHb-like globins are closely related to bacterial Hbs. Figure 2 also shows that Micropusil, Microsp, and Ostreosp nsHbs are closer to bryophyte than to other land plant nsHbs, and thus are in an ancestral position to land plant nsHbs. Nucleotide analysis revealed that Micropusil, Microsp, and Ostreosp nshb-like genes lack introns, whereas all known land plant nshb genes are interrupted by three introns [7, 19, 39, 40]. Thus, if the Micromonas and Ostreococcus nsHb-like globins were ancestors to land plant nsHbs, we speculate that introns inserted into an algal nshb-like gene prior to the origin of land plant nshbs to evolve into a conserved 4 exons/3 introns gene structure. 3.3. In silico characterization of Micromonas and Ostreococcus nsHb-like globin sequences

Further in silico analysis showed that predicted molecular weight for Micropusil, Microsp, and Ostreosp nsHbs is 13 857, 16 403, and 17 156 Da, respectively, which is within the range for known land plant nsHbs and Lbs [7, 19, 41]. Also, no leader peptides and targeting sequences for chloroplast and mitochondria translocation were detected in the Micropusil, Microsp, and Ostreosp nsHb sequences. These observations suggest that Micromonas and Ostreococcus nsHb-like globins are located in vivo in the cell cytoplasm. Hydropathy analysis showed that profiles for Micropusil, Microsp, and Ostreosp nsHbs and rice Hb1 are highly similar (Figure. 3). This evidence suggests that Micromonas and Ostreococcus nsHb-like globins fold similarly to rice Hb1 (below). However, position ~20-40 is more hydrophobic in Microsp nsHb than in Micropusil and Ostreosp nsHbs and rice Hb1. This indicates that regions within helices A and B are highly hydrophobic in Microsp nsHb compared to those in Micropusil and Ostreosp nsHbs and rice Hb1.



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Table 1. Highest E value and sequence similarity and identity of prasinophyceae (Micromonas and Ostreococcus) nsHb-like globins and sequences deposited in the Genbank database. Similarity and identity values were identified after BLASTp of Micromonas pusilla, Micromonas sp. RCC299, and Ostreococcus sp. RCC809 nsHb sequences (Figure 1) with sequences deposited in the Genbank database. Similarity values show amino acid position with identical polarity (negative, positive, or nonpolar) in aligned sequences. Identity values show identical amino acids in aligned sequences. Land plant nsHb entries are indicated with an asterisk. Prasinophyceae Highest hits GenBank E value Identity Similarity (nsHb-like) sequence entry (%) (%) Micromonas pusilla nsHb Sorangium cellulosum Hb YP_001611205.1 1e-17 40 53 Rhodopsedumonas palustris Hb YP_780194.1 1e-17 42 56 Strongylocentrotus purpuratus Hb XP_001199205.1 6e-17 35 50 Bradyrhizobium sp. Hb YP_001238856.1 7e-17 39 52 Xanthobacter autotrophicus Hb YP_001417400.1 5e-16 42 55 *Zea mays Hb NP_001104966.1 1e-15 39 54 Shewanella amazonensis Hb YP_929350.1 1e-15 38 50 Nitrobacter sp. Hb ZP_01047463.1 1e-15 40 56 *Lotus japonicus nsHb dbj|BAE46739.1 1e-15 35 53 Methylacidiphilum infernorum Hb YP_001939748.1 2e-15 32 56 *Glycine max nsHb AAA97887 2e-25 36 52 *Marchantia polymorpha Hb AAK07743 4e-15 42 55 *Arabidopsis thaliana AHb2 NP_187663.1 4e-15 36 54 *Brassica napus nsHb-2 AAK07741 5e-15 35 53 Micromonas sp. RCC299 nsHb Strongylocentrotus ourouratus Hb XP_001199205.1 5e-19 39 55 Methylobacterium sp. Hb YP_001767602.1 5e-19 38 57 Verrucomicrobiae bacterium Hb ZP_05057247.1 1e-17 35 52 Bradyrhizobium sp. Hb YP_001238856.1 5e-17 37 55 Rhodopseudomonas palustris Hb YP_485375.1 8e-17 35 52 Azorhizobium caulinodans Hb YP_001523599.1 1e-16 40 52 Xanthobacter autotrophicus Hb YP_001417400.1 3e-16 37 51 Shewanella woodyi Hb YP_001758583.1 3e-16 37 53 Gemmatimonas aurantiaca Hb YP_002763275.1 3e-16 34 49 Sorangium cellulosum Hb YP_001611205.1 6e-16 38 53



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Rhodopirellula baltica Hb NP_864649.1 4e-15 34 50 Bradyrhizobium japonicum Hb NP_769447.1 1e-14 32 52 Rhodobacterales bacterium Hb ZP_01012339.1 2e-14 35 48 Ralstonia eutropha Hb YP_729107.1 3e-14 35 49 Thalassiosira pseudonana Hb XP_002291928.1 5e-14 34 51 *Brassica napus nsHb-2 AAK07741 9e-14 32 55 *Arabidopsis thaliana nsHb-2 NP_187663.1 2e-13 33 56 *Medicago sativa Lb AAA32659 7e-13 34 56 *Cichorium intibus x C. endivia nsHb CAA07547 9e-13 32 55 Ostreococcus sp. RCC809 nsHb Sorangium cellulosum Hb YP_001611205.1 5e-22 43 57 Rhodopseudomonas palustris Hb YP_780194.1 3e-20 44 58 Xanthobacter autotrophicus Hb YP_001417400.1 3e-18 38 52 Bradyrhizobium sp. Hb YP_001204533.1 3e-18 39 54 Verrucomicrobiae bacterium Hb ZP_05057247.1 5e-18 39 56 Azorhizobium culinodans Hb YP_001523599.1 8e-18 38 53 Rattus norvegicus Ngb NP_203523.2 2e-17 36 53 Nitrobacter sp. Hb ZP_01047463.1 3e-17 38 55 Gemmatimonas aurantiaca Hb YP_002763275.1 4e-17 37 52 Canis lupus familiaris Ngb NP_001003356.2 4e-17 39 54 Methylobacterium sp. Hb YP_001767602.1 4e-17 42 58 Strongylocentrotus purpuratus Hb XP_001199205.1 5e-17 32 54 Homo sapiens Ngb EAW81275 5e-17 36 52 Equus caballus Ngb XP_001493389.1 6e-17 35 53 Rhodopsedudomonas palustris Hb YP_485375.1 6e-17 38 52 Mus musculus Ngb NP_071859.1 8e-17 35 53 *Marchantia polymorpha nsHb AAK07743 8e-17 43 60 Spalax galili Ngb CAL91961 1e-16 35 53 Macaca mulatta Ngb NP_001030591.1 1e-16 35 51 (several other Ngbs and few bacterial globins) *Oryza sativa nsHb1 NP_001049476.1 3e-15 38 57 *Lotus japonicus nsHb BAE46739 8e-15 35 52 *Zea mays nsHb NP_001104966.1 2e-14 37 55



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Figure 1 (To be continued on next page). Sequence alignment of Micromonas and Ostreococcus nsHb-like globins with selected bacterial Hbs and land plant nsHbs and Lbs. ,distal amino acid (His/Gln) and proximal His; *, Phe B10 and CD1; black background, postulated amino acids for the dimer interface in the rice Hb1 structure [38]; gray background, highly conserved amino acids. Helix position corresponds to helices from the rice Hb1 structure [38]. Amino acid sequences were obtained from the Genbank database using the following accession numbers: AAG01375 (Zea mays Hb), AAC49882 (Oryza sativa Hb1), AAB82769 (Arabidopsis thaliana AHb1), AAA97887 (Glycine max Hb), ABR68293 (Chamaecrista fasciculata Hb), AAA86756 (Vigna unguiculata LbI), CAA23731 (Glycine max Lba), AAB82770 (Arabidopsis thaliana AHb2), ABK20873 (Physcomitrella patens Hb), ABK41124 (Ceratodon purpureus Hb), AAK07743 (Marchantia polymorpha Hb), YP_780194.1 (Rodopseudomonas palustris Hb), YP_001204533.1 (Bradyrhizobium sp. Hb), and YP_001417400.1 (Xanthobacter autotrophicus Hb).

100

150



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Figure 1 (Continued)

Figure 2. Cluster analysis of Micromonas and Ostreococcus nsHb-like globins and selected bacterial Hbs and land plant nsHbs and Lbs. The dendrogram was constructed from Hb sequences aligned in figure 1 using the Neighbor Joining method of the Clustal X program.



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Micropusil nsHb Microsp nsHb

Ostreosp nsHb Rice Hb1

Figure 3. Hydropathy analysis of Micromonas and Ostreococcus nsHb-like globin and rice Hb1 sequences. For experimental details see the Experimental section.



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3.4. Modeling and analysis of Micromonas and Ostreococcus nsHb-like globins tertiary structure

Because of the high similarity among the hydropathic profiles of Micropusil, Microsp, and Ostreosp nsHbs and rice Hb1, the tertiary structure of Micromonas and Ostreococcus nsHb-like globins was modeled and models were compared to the crystal structure of rice Hb1 (PDB ID 1D8U) [38]. Models were obtained using the automated mode of the Swiss Model. In this mode, the structure of Micropusil and Ostreosp nsHbs and Microsp nsHb was predicted from template Alcaligenes eutrophus fHb (PDB ID 1CQX) and soybean Lba (PDB ID 1BIN) crystal structures, respectively. The sequence identity and E-value for the Micropusil nsHb-A. eutrophus fHb, Microsp nsHb-soybean Lba, and Ostreosp nsHb-A. eutrophus fHb pair of aligned sequences were 26.6% and 3.6e-25, 25.7% and

7.3e-24, and 21.9% and 5e-27, respectively. Evaluation using the Verify3D option from the Swiss Model indicated that models for predicted Micropusil, Microsp, and Ostreosp nsHbs are reliable, as values along each sequence were higher than 0.

Figure 4 shows that the predicted structure of Micromonas and Ostreococcus nsHb-like globins is highly similar to that of native rice Hb1, including the positions of helices E and F, where distal and proximal His are located, respectively. This observation shows that Micromonas and Ostreococcus nsHb-like globins could fold into the canonical Mb-fold. Minor differences between the predicted structure of Micromonas and Ostreococcus nsHb-like globins and that of native rice Hb1 were detected at the length of the pre-helix A/helix A and C-terminal, and at the CD-, EF-, and GH-loops. A distinctive characteristic for the

Figure 4. Overlay of predicted Micromonas and Ostreococcus nsHb-like globins (blue) and native rice Hb1 (yellow) tertiary structures. Helices (including to prehelix A) are indicated with letters A-H. The predicted structure of Micromonas and Ostreococcus nsHb-like globins is deposited in the Protein Model Database (http://mi.caspur.it/PMDB/, last accessed on December 2009) under the ID number PM0076078, PM0076079, and PM0076080, respectively. For experimental details see the Experimental section.



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predicted structure of Ostreosp nsHb was the existence of -sheet foldings within the CD-loop region, which is atypical of the Mb-fold.

An examination of the amino acids that are essential for binding of ligands to the heme-Fe showed that the position and distance of Phe B10 and CD1 and proximal His are similar in predicted Micromonas and Ostreococcus nsHb-like globins and native rice Hb1 (Figure 5, Table 2). Distal His is located similarly in predicted Microsp nsHb and native rice Hb1, which

suggests that heme-Fe in Microsp nsHb is hexacoordinate. In contrast, distance of distal His in predicted Micropusil and Ostreosp nsHbs is ~10 and 8 Å farther from the heme-Fe than in native rice Hb1 (Table 2). This observation suggests that heme-Fe in Micropusil and Ostreosp nsHbs is pentacoordinate. However, the above observations should be further verified by experimental analysis, such as that from either x-ray crystallography or NMR of recombinant proteins.

Figure 5. Overlay of selected amino acids in the predicted Micromonas and Ostreococcus nsHb-like globins (blue) and native rice Hb1 (yellow) heme pocket. For experimental details see the Experimental section.



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4. Conclusions

Hemoglobins are widely distributed in living organisms, from bacteria to mammals, including plants [16, 42, 43]. Although horizontal gene transfer of hbs has been identified in diverse organisms [16, 44], results from this work suggest that land plant nsHbs evolved from algal nsHb-like globins, such as those from the Micromonas and Ostreococcus nsHb-like globins.

A number of structural characteristics in land plant nsHbs were identified in the Micromonas and Ostreococcus nsHb-like globins, such as the existence of Phe B10 and CD1 and proximal and distal His. Also, predicted Micromonas and Ostreococcus nsHb-like globins fold into the Mb-fold. These observations indicate that the major structural properties of plant nsHbs originated more than 850 MYA and were conserved during evolution to maintain plant function and adaptation. Acknowledgements

Authors wish to express their gratitude to Miss Gillian Klucas for corrections made to improve the English language. Work in R.A.-P´s.

laboratory has been financed by SEP-PROMEP (grant number UAEMor-PTC-01-01/PTC23) and Consejo Nacional de Ciencia y Tecnología

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Table 2. Distance of amino acid residues that either interact with Fe at the heme prosthetic group or affect binding of ligands to Fe in predicted Micromonas and Ostreococcus nsHbs-like globins and native rice Hb1.

Distance to heme-Fe (Å) Globin Phe B10 Phe CD1 Proximal His Distal His Micropusil nsHb 10.15 5.79 2.20 12.53 Microsp nsHb 9.26 5.81 1.99 4.47 Ostreosp nsHb 9.19 6.54 2.84 10.81 Rice Hb1 7.21 5.45 2.11 2.04



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