Raúl Arredondo-Peter

Crystal structure of a nonsymbiotic plant hemoglobin.

BACKGROUND Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.

Rice hemoglobins. Gene cloning, analysis, and O2-binding kinetics of a recombinant protein synthesized in Escherichia coli.

Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.

The evolution of land plant hemoglobins

This review discusses the evolution of land plant hemoglobins within the broader context of eukaryote hemoglobins and the three families of bacterial globins. Most eukaryote hemoglobins, including metazoan globins and the symbiotic and non-symbiotic plant hemoglobins, are homologous to the bacterial 3/3-fold flavohemoglobins. The remaining plant hemoglobins are homologous to the bacterial 2/2-fold group 2 hemoglobins. We have proposed that all eukaryote globins were acquired via horizontal gene transfer concomitant with the endosymbiotic events responsible for the origin of mitochondria and chloroplasts. Although the 3/3 hemoglobins originated in the ancestor of green algae and plants prior to the emergence of embryophytes at about 450 mya, the 2/2 hemoglobins appear to have originated via horizontal gene transfer from a bacterium ancestral to present day Chloroflexi. Unlike the 2/2 hemoglobins, the evolution of the 3/3 hemoglobins was accompanied by duplication, diversification, and functional adaptations. Duplication of the ancestral plant nshb gene into the nshb-1 and nshb-2 lineages occurred prior to the monocot-dicot divergence at ca. 140 mya. It was followed by the emergence of symbiotic hemoglobins from a non-symbiotic hemoglobin precursor and further specialization, leading to leghemoglobins in N₂-fixing legume nodules concomitant with the origin of nodulation at ca. 60 mya. The transition of non-symbiotic to symbiotic hemoglobins (including to leghemoglobins) was accompanied by the alteration of heme-Fe coordination from hexa- to penta-coordination. Additional genomic information about Charophyte algae, the sister group to land plants, is required for the further clarification of plant globin phylogeny.

Phylogenetic Relationships of 3/3 and 2/2 Hemoglobins in Archaeplastida Genomes to Bacterial and Other Eukaryote Hemoglobins

Land plants and algae form a supergroup, the Archaeplastida, believed to be monophyletic. We report the results of an analysis of the phylogeny of putative globins in the currently available genomes to bacterial and other eukaryote hemoglobins (Hbs). Archaeplastida genomes have 3/3 and 2/2 Hbs, with the land plant genomes having group 2 2/2 Hbs, except for the unexpected occurrence of two group 1 2/2 Hbs in Ricinus communis. Bayesian analysis shows that plant 3/3 Hbs are related to vertebrate neuroglobins and bacterial flavohemoglobins (FHbs). We sought to define the bacterial groups, whose ancestors shared the precursors of Archaeplastida Hbs, via Bayesian and neighbor-joining analyses based on COBALT alignment of representative sets of bacterial 3/3 FHb-like globins and group 1 and 2 2/2 Hbs with the corresponding Archaeplastida Hbs. The results suggest that the Archaeplastida 3/3 and group 1 2/2 Hbs could have originated from the horizontal gene transfers (HGTs) that accompanied the two generally accepted endosymbioses of a proteobacterium and a cyanobacterium with a eukaryote ancestor. In contrast, the origin of the group 2 2/2 Hbs unexpectedly appears to involve HGT from a bacterium ancestral to Chloroflexi, Deinococcales, Bacilli, and Actinomycetes. Furthermore, although intron positions and phases are mostly conserved among the land plant 3/3 and 2/2 globin genes, introns are absent in the algal 3/3 genes and intron positions and phases are highly variable in their 2/2 genes. Thus, introns are irrelevant to globin evolution in Archaeplastida.

Cloning and characterization of a caesalpinoid (Chamaecrista fasciculata) hemoglobin: The structural transition from a nonsymbiotic hemoglobin to a leghemoglobin

Nonsymbiotic hemoglobins (nsHbs) and leghemoglobins (Lbs) are plant proteins that can reversibly bind O2 and other ligands. The nsHbs are hexacoordinate and appear to modulate cellular concentrations of NO and maintain energy levels under hypoxic conditions. The Lbs are pentacoordinate and facilitate the diffusion of O2 to symbiotic bacteroids within legume root nodules. Multiple lines of evidence suggest that all plant Hbs evolved from a common ancestor and that Lbs originated from nsHbs. However, little is known about the structural intermediates that occurred during the evolution of pentacoordinate Lbs from hexacoordinate nsHbs. We have cloned and characterized a Hb (ppHb) from the root nodules of the ancient caesalpinoid legume Chamaecrista fasciculata. Protein sequence, modeling data, and spectral analysis indicated that the properties of ppHb are intermediate between that of nsHb and Lb, suggesting that ppHb resembles a putative ancestral Lb. Predicted structural changes that appear to have occurred during the nsHb to Lb transition were a compaction of the CD‐loop and decreased mobility of the distal His inhibiting its ability to coordinate directly with the heme‐Fe, leading to a pentacoordinate protein. Other predicted changes include shortening of the N‐ and C‐termini, compaction of the protein into a globular structure, disappearance of positive charges outside the heme pocket and appearance of negative charges in an area located between the N‐ and C‐termini. A major consequence for some of these changes appears to be the decrease in O2‐affinity of ancestral nsHb, which resulted in the origin of the symbiotic function of Lbs. Proteins 2008.

Molecular Cloning and Characterization of a Moss (Ceratodon purpureus) Nonsymbiotic Hemoglobin Provides Insight into the Early Evolution of Plant Nonsymbiotic Hemoglobins

Nonsymbiotic hemoglobins (nsHbs) are widespread in plants including bryophytes. Bryophytes (such as mosses) are among the oldest land plants, thus an analysis of a bryophyte nsHb is of interest from an evolutionary perspective. However, very little is known about bryophyte nsHbs. Here, we report the cloning and characterization of an nshb gene (cerhb) from the moss Ceratodon purpureus. Sequence analysis showed that cerhb is interrupted by 3 introns in identical position as all known plant nshb genes, which suggests that the ancestral nshb gene was interrupted by 3 introns. Expression analysis showed that cerhb expresses in protonemas and gametophytes growing in normal conditions and that it overexpresses in protonemas subjected to osmotic (sucrose), heat-shock, cold-, and nitrate-stress conditions. Also, modeling of the Ceratodon nsHb (CerHb) tertiary structure suggests that CerHb is hexacoordinate and that it binds O(2) with high affinity. Comparative analysis of the predicted CerHb with native rice Hb1 and soybean leghemoglobin a structures revealed that the major evolutionary changes that probably occurred during the evolution of plant Hbs were 1) a hexacoordinate to pentacoordinate transition at the heme prosthetic group, 2) a length decrease at the CD-loop and N- and C-termini regions, and 3) the compaction of the protein into a globular structure.

Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli : Purification and characterization of the recombinant protein

Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII and LbIII) that are encoded by at least two genes. We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR. Primers were designed using the sequence of the soybean lbc gene. The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids. Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli. Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques. Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques. The data showed that the structural and spectral characteristics of rLbII and nLbII were similar. The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H2O2 was 86-times lower than that of HRP (K(m)=23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H2O2 as electron donor was 2838-times lower than that of HRP (k(cat)/K(m)=15.8 and 44,833 mM(-1) min(-1), respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H2O2 concentration, and that it poorly protects recombinant Escherichia coli from H2O2 stress. These observations indicate that rice Hb1 inefficiently scavenges H2O2 as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.

What are the origins and phylogeny of plant hemoglobins

Land plants and algae are now represented by about 40 genomes. Although most are incomplete, putative globins appear to be present in all the ca. 30 land plant genomes and in all except one algal genomes. The globins have either the canonical 3/3 α-helical fold characteristic of vertebrate myoglobin (Mb) or 2/2 α-helical folds, characteristic of bacterial globins with a truncated Mb-fold. In view of the fairly complete picture of the globin superfamily that is now available from analyses of over 1000 bacterial genomes and >200 other eukaryote genomes, it is now possible to seek answers to the following twin questions: what is the phylogenetic relationship of plant and algal globins to those of other eukaryotes and what is their likely bacterial origin? We summarize below the available results. Molecular phylogenetic analyses indicate that plant and algal 3/3 globins are related to bacterial flavohemoglobins and vertebrate neuroglobins. Furthermore, they also suggest that plant and algal 3/3 and group 1 2/2 Hbs originated from the horizontal gene transfers that accompanied the two generally accepted endosymbioses of a proteobacterium and a cyanobacterium with a eukaryote ancestor. In contrast, the origin of the group 2 2/2 Hbs unexpectedly appears to involve horizontal gene transfer from a bacterium ancestral to Chloroflexi, Deinococcales, Bacillli and Actinomycetes. We present additional results which indicate that the shared ancestry is likely to be with the Chloroflexi alone.

Prediction of folding pathway and kinetics among plant hemoglobins using an average distance map method

Computational methods, such as the ADM (average distance map) method, have been developed to predict folding of homologous proteins. In this work we used the ADM method to predict the folding pathway and kinetics among selected plant nonsymbiotic (nsHb), symbiotic (Lb), and truncated (tHb) hemoglobins (Hbs). Results predicted that (1) folding of plant Hbs occurs throughout the formation of compact folding modules mostly formed by helices A, B, and C, and E, F, G, and H (folding modules A/C and E/H, respectively), and (2) primitive (moss) nsHbs fold in the C→N direction, evolved (monocot and dicot) nsHbs fold either in the C→N or N→C direction, and Lbs and plant tHbs fold in the C→N direction. We also predicted relative folding rates of plant Hbs from qualitative analyses of the stability of subdomains and classified plant Hbs into fast and moderate folding. ADM analysis of nsHbs predicted that prehelix A plays a role during folding of the N‐terminal domain of Ceratodon nsHb, and that CD‐loop plays a role in folding of primitive (Physcomitrella and Ceratodon) but not evolved nsHbs. Modeling of the rice Hb1 A/C and E/H modules showed that module E/H overlaps to the Mycobacterium tuberculosis HbO two‐on‐two folding. This observation suggests that module E/H is an ancient tertiary structure in plant Hbs. Proteins 2005.