Refractory severe idiopathic thrombocytopenia and treatment challenges.

Refractory idiopathic thrombocytopenia (ITP) is a disease that does not respond to or relapses after splenectomy, requires treatment to reduce the risk of clinically significant bleeding, and is a challenging case to treat. Presentation of the Case: A 39-year-old male with a history of chronic ITP presented with a platelet count of 1000/µl and prostatitis. He was started on Ciprofloxacin and started intravenous immunoglobulin along with intravenous methylprednisolone. Then Rituximab was started on day fourth. Since his platelet remained 0/µl, Mycophenolate mofetil (Cellcept) was started on day 14th. Next, a dose of Romiplostim on day 19th was given. Eltrombopag (Promacta) and Tavlesse were started on day 23th and platelets rose to 96×103/µl on day 26th and then 418×103/µl. Discussion: Normally, refractory ITP patients who do not respond to first-line treatments require a combination therapy of one to two medicines of the second line, like thrombopoietin receptor agonists. However, this patient's thrombocytopenia neither responded to first-line treatment nor second-line treatment with Promacta/Romiplostin plus immunosuppressives or Tavlesse. Conclusion: Refractory ITP, who has not responded to first-line and second-line treatments, requires treatment with a combination of all first-line and second-line treatments. Furthermore, Promacta, Tavlesse, and Romiplostim have a big role to play in helping the patient.

Oxygen Generation via Water Splitting by a Novel Biogenic Metal Ion-Binding Compound.

Methanobactins (MBs) are small (<1,300-Da) posttranslationally modified copper-binding peptides and represent the extracellular component of a copper acquisition system in some methanotrophs. Interestingly, MBs can bind a range of metal ions, with some being reduced after binding, e.g., Cu2+ reduced to Cu+. Other metal ions, however, are bound but not reduced, e.g., K+. The source of electrons for selective metal ion reduction has been speculated to be water but never empirically shown. Here, using H218O, we show that when MBs from Methylocystis sp. strain SB2 (MB-SB2) and Methylosinus trichosporium OB3b (MB-OB3) were incubated in the presence of either Au3+, Cu2, or Ag+, 18,18O2 and free protons were released. No 18,18O2 production was observed in the presence of either MB-SB2 or MB-OB3b alone, gold alone, copper alone, or silver alone or when K+ or Mo2+ was incubated with MB-SB2. In contrast to MB-OB3b, MB-SB2 binds Fe3+ with an N2S2 coordination and will also reduce Fe3+ to Fe2+. Iron reduction was also found to be coupled to the oxidation of 2H2O and the generation of O2. MB-SB2 will also couple Hg2+, Ni2+, and Co2+ reduction to the oxidation of 2H2O and the generation of O2, but MB-OB3b will not, ostensibly as MB-OB3b binds but does not reduce these metal ions. To determine if the O2 generated during metal ion reduction by MB could be coupled to methane oxidation, 13CH4 oxidation by Methylosinus trichosporium OB3b was monitored under anoxic conditions. The results demonstrate that O2 generation from metal ion reduction by MB-OB3b can support methane oxidation. IMPORTANCE The discovery that MB will couple the oxidation of H2O to metal ion reduction and the release of O2 suggests that methanotrophs expressing MB may be able to maintain their activity under hypoxic/anoxic conditions through the "self-generation" of dioxygen required for the initial oxidation of methane to methanol. Such an ability may be an important factor in enabling methanotrophs to not only colonize the oxic-anoxic interface where methane concentrations are highest but also tolerate significant temporal fluctuations of this interface. Given that genomic surveys often show evidence of aerobic methanotrophs within anoxic zones, the ability to express MB (and thereby generate dioxygen) may be an important parameter in facilitating their ability to remove methane, a potent greenhouse gas, before it enters the atmosphere.

Plant hemoglobins: a journey from unicellular green algae to vascular plants.

Globins (Glbs) are widely distributed in archaea, bacteria and eukaryotes. They can be classified into proteins with 2/2 or 3/3 α-helical folding around the heme cavity. Both types of Glbs occur in green algae, bryophytes and vascular plants. The Glbs of angiosperms have been more intensively studied, and several protein structures have been solved. They can be hexacoordinate or pentacoordinate, depending on whether a histidine is coordinating or not at the sixth position of the iron atom. The 3/3 Glbs of class 1 and the 2/2 Glbs (also called class 3 in plants) are present in all angiosperms, whereas the 3/3 Glbs of class 2 have been only found in early angiosperms and eudicots. The three Glb classes are expected to play different roles. Class 1 Glbs are involved in hypoxia responses and modulate NO concentration, which may explain their roles in plant morphogenesis, hormone signaling, cell fate determination, nutrient deficiency, nitrogen metabolism and plant-microorganism symbioses. Symbiotic Glbs derive from class 1 or class 2 Glbs and transport O2 in nodules. The physiological roles of class 2 and class 3 Glbs are poorly defined but could involve O2 and NO transport and/or metabolism, respectively. More research is warranted on these intriguing proteins to determine their non-redundant functions.

Effect of curcumin supplementation on serum expression of select cytokines and chemokines in a female rat model of nonalcoholic steatohepatitis.

OBJECTIVE: We recently reported that curcumin supplementation in a metabolically (i.e., Western diet [WD]) and chemically (i.e., CCl4) induced female rat model of non-alcoholic steatohepatitis (NASH) was associated with lower liver pathology scores and molecular markers of inflammation. This occurred when curcumin was given during induction of disease (preventative arm; 8-week WD with or without curcumin [8WD + C vs. 8WD]) as well as when given after disease development (treatment arm; 12-week WD with or without curcumin during weeks 9-12 [12WD + C vs. 12WD]). Herein, we sought to extend our findings from that study by determining the effects of curcumin supplementation on cytokine/chemokine expression in serum collected from these same rats. RESULTS: 24 cytokines/chemokines were assayed. IL-2 (+ 80%) and IL-13 (+ 83%) were greater with curcumin supplementation in the prevention arm. IL-2 (+ 192%), IL-13 (+ 87%), IL-17A (+ 81%) and fractalkine (+ 121%) were higher while RANTES was lower (- 22%) with curcumin supplementation in the treatment arm (p < 0.05 for all). RANTES concentrations also correlated significantly with hepatic pathology scores of inflammation (r = 0.417, p = 0.008). Select serum cytokines/chemokines were affected with curcumin supplementation in this female rat model of NASH. Moreover, curcumin's effect(s) on RANTES and its association with liver disease pathogenesis and progression may warrant further investigation.

The role of the NADH-dependent nitrite reductase, Nir, from Escherichia coli in fermentative ammonification.

Nitrate and nitrite reduction are of paramount importance for nitrogen assimilation and anaerobic metabolism, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates cellular responses to their environments. The soluble, cytoplasmic siroheme NADH-nitrite reductase (Nir) in Escherichia coli is necessary for nitrate/nitrite assimilation but has also been reported to either "detoxify" nitrite, or to carry out fermentative ammonification in support of anaerobic catabolism. Theoretically, nitrite detoxification would be important for anaerobic growth on nitrate, during which excess nitrite would be reduced to ammonium. Fermentative ammonification by Nir would be important for maximization of non-respiratory ATP production during anaerobic growth in the presence of nitrite. Experiments reported here were designed to test the potential role of Nir in fermentative ammonification directly by growing E. coli along with mutant strains lacking Nir or the respiratory nitrite reductase (Nrf) under anaerobic conditions in defined media while monitoring nitrogen utilization and fermentation metabolites. To focus on the role of Nir in fermentative ammonification, pH control was used in most experiments to eliminate nitrite toxicity due to nitric acid formation. Our results demonstrate that Nir confers a significant benefit during fermentative growth that reflects fermentative ammonification rather than detoxification. We conclude that fermentative ammonification by Nir allows for the energetically favorable fermentation of glucose to formate and acetate. These results and conclusions are discussed in light of the roles of Nir in other bacteria and in plants.

Spectroscopic evidence supporting neutral thiol ligation to ferrous heme iron.

The binding of neutral thiol (ethanethiol, EtSH) or thioether (tetrahydrothiophene, THT) to two types of heme proteins in their ferrous state has been investigated with UV-visible (UV-Vis) absorption and magnetic circular dichroism spectroscopy. For the second GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain from the sensory kinase MsmS (sGAF2), stepwise additions of these respective two sulfur-donor ligands to its dithionite-reduced ferrous form generate homogeneous six-coordinate low-spin ferrous complexes at both pHs 7.0 and 5.4. Similar complexes were partially formed for deoxyferrous soybean leghemoglobin with EtSH or THT within their solubility limits in water. The titrations cause significant UV-Vis spectra changes attributable to a five-coordinate to six-coordinate heme iron coordination change. For sGAF2, the resulting spectra are essentially identical for the both ligands, clearly indicating the direct binding of neutral thiol/thioether to ferrous heme iron as the distal ligand. On the other hand, the thiol EtSH binds to ferric sGAF2 in the anionic thiolate form, while thioether THT forms its ferric sGAF2 complex as a neutral ligand. These observations provide compelling evidence that neutral cysteine is a plausible ligand for ferrous heme proteins.

Steady-State Kinetics of Phytoglobin-Catalyzed Reduction of Hydroxylamine to Ammonium.

Phytoglobins are plant hexacoordinate hemoglobins with reversible coordination of a histidine side chain to the ligand binding site of the heme iron. They mediate electron transfer reactions such as nitric oxide scavenging and are particularly efficient at reducing nitrite and hydroxylamine. Previous work with phytoglobins has focused only on single turnovers of these reactions and has not revealed whether structural features, such as histidine hexacoordination, play a prominent role in the complete catalytic cycle. This work characterizes steady-state phytoglobin catalysis of reduction of hydroxylamine to ammonium using two different chemical reductants. Km and kcat values were measured for rice phytoglobin, horse myoglobin, human neuroglobin, and a rice phytoglobin mutant protein in which the hexacoordinating histidine has been replaced with leucine (Phyt:H73L). The results demonstrate that phytoglobin catalysis driven by benzyl viologen is limited only by the dissociation rate constant for the distal histidine. This is consistent with the rate limit in single-turnover experiments and demonstrates that the kinetics of hydroxylamine binding, and not phytoglobin reduction, ultimately governs the reaction. Catalysis by the other proteins that either lack or have tighter hexacoordination is much slower, suggesting that facile reversibility of the bond between the distal histidine and the heme iron is needed to allow both substrate binding and heme iron reduction. On the other hand, catalysis driven by dithionite is limited by SO2•- concentrations and is similar for all of these proteins, suggesting that dithionite is not a good reducing agent for evaluation of the catalytic properties of hemoglobins.

Role of Reversible Histidine Coordination in Hydroxylamine Reduction by Plant Hemoglobins (Phytoglobins).

Reduction of hydroxylamine to ammonium by phytoglobin, a plant hexacoordinate hemoglobin, is much faster than that of other hexacoordinate hemoglobins or pentacoordinate hemoglobins such as myoglobin, leghemoglobin, and red blood cell hemoglobin. The reason for differences in reactivity is not known but could be intermolecular electron transfer between protein molecules in support of the required two-electron reduction, hydroxylamine binding, or active site architecture favoring the reaction. Experiments were conducted with phytoglobins from rice, tomato, and soybean along with human neuroglobin and soybean leghemoglobin that reveal hydroxylamine binding as the rate-limiting step. For hexacoordinate hemoglobins, binding is limited by the dissociation rate constant for the distal histidine, while leghemoglobin is limited by an intrinsically low affinity for hydroxylamine. When the distal histidine is removed from rice phytoglobin, a hydroxylamine-bound intermediate is formed and the reaction rate is diminished, indicating that the distal histidine imidazole side chain is critical for the reaction, albeit not for electron transfer but rather for direct interaction with the substrate. Together, these results demonstrate that phytoglobins are superior at hydroxylamine reduction because they have distal histidine coordination affinity constants near 1, and facile rate constants for binding and dissociation of the histidine side chain. Hexacoordinate hemoglobins such as neuroglobin are limited by tighter histidine coordination that blocks hydroxylamine binding, and pentacoordinate hemoglobins have intrinsically lower hydroxylamine affinities.

Phytoglobin: a novel nomenclature for plant globins accepted by the globin community at the 2014 XVIII conference on Oxygen-Binding and Sensing Proteins.

Hemoglobin (Hb) is a heme-containing protein found in the red blood cells of vertebrates. For many years, the only known Hb-like molecule in plants was leghemoglobin (Lb). The discovery that other Hb-like proteins existed in plants led to the term "nonsymbiotic Hbs (nsHbs)" to differentiate them from the Lbs. While this terminology was adequate in the early stages of research on the protein, the complexity of the research in this area necessitates a change in the definition of these proteins to delineate them from red blood cell Hb. At the 2014 XVIII Conference on Oxygen-Binding and Sensing Proteins, the group devoted to the study of heme-containing proteins, this issue was discussed and a consensus was reached on a proposed name change. We propose Phytoglobin (Phytogb) as a logical, descriptive name to describe a heme-containing (Hb-like) protein found in plants. It will be readily recognized by the research community without a prolonged explanation of the origin of the term. The classification system that has been established can essentially remain unchanged substituting Phytogb in place of nsHb. Here, we present a guide to the new nomenclature, with reference to the existing terminology and a phylogenetic scheme, placing the known Phytogbs in the new nomenclature.

Electron self-exchange in hemoglobins revealed by deutero-hemin substitution.

Hemoglobins (phytoglobins) from rice plants (nsHb1) and from the cyanobacterium Synechocystis (PCC 6803) (SynHb) can reduce hydroxylamine with two electrons to form ammonium. The reaction requires intermolecular electron transfer between protein molecules, and rapid electron self-exchange might play a role in distinguishing these hemoglobins from others with slower reaction rates, such as myoglobin. A relatively rapid electron self-exchange rate constant has been measured for SynHb by NMR, but the rate constant for myoglobin is equivocal and a value for nsHb1 has not yet been measured. Here we report electron self-exchange rate constants for nsHb1 and Mb as a test of their role in hydroxylamine reduction. These proteins are not suitable for analysis by NMR ZZ exchange, so a method was developed that uses cross-reactions between each hemoglobin and its deutero-hemin substituted counterpart. The resulting electron transfer is between identical proteins with low driving forces and thus closely approximates true electron self-exchange. The reactions can be monitored spectrally due to the distinct spectra of the prosthetic groups, and from this electron self-exchange rate constants of 880 (SynHb), 2900 (nsHb1), and 0.05M(-1) s(-1) (Mb) have been measured for each hemoglobin. Calculations of cross-reactions using these values accurately predict hydroxylamine reduction rates for each protein, suggesting that electron self-exchange plays an important role in the reaction.

Nitric oxide in plants: the roles of ascorbate and hemoglobin.

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.

Switchgrass PviCAD1: understanding residues important for substrate preferences and activity.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis. Although plants contain numerous genes coding for CADs, only one or two CADs appear to have a primary physiological role in lignin biosynthesis. Much of this distinction appears to reside in a few key residues that permit reasonable catalytic rates on monolignal substrates. Here, several mutant proteins were generated using switchgrass wild type (WT) PviCAD1 as a template to understand the role of some of these key residues, including a proton shuttling HL duo in the active site. Mutated proteins displayed lowered or limited activity on cinnamylaldehydes and exhibited altered kinetic properties compared to the WT enzyme, suggesting that key residues important for efficient catalysis had been identified. We have also shown that a sorghum ortholog containing EW, instead of HL in its active site, displayed negligible activity against monolignals. These results indicate that lignifying CADs require a specific set of key residues for efficient activity against monolignals.

Spectral and copper binding properties of methanobactin from the facultative methanotroph Methylocystis strain SB2.

Methanobactin (mb) is the first characterized example of a chalkophore, a class of copper-binding chromopeptides similar to iron-binding siderophores. Structural, redox, themodynamic, and spectral studies on chalkophores have focused almost exclusively on the mb from Methylosinus trichosporium OB3b (mb-OB3b). The structural characterization of a second mb from Methylocystis strain SB2 (mb-SB2) provides a means to examine the core structural features and metal binding properties of this group of chromopeptides. With the exception of the 5-membered rings (either oxazolone or imidazolone), enethiol groups, and the N-terminus oxo group, the structure of mb-SB2 differs markedly from mb-OB3b. In particular the amino acids commonly associated with metal coordination and redox activity found in mb-OB3b, Cys, Met, and Try, are replaced by Ala or are missing in mb-SB2. In this report the spectral and thermodynamic properties of mb-SB2 are presented and compared to mb-OB3b. The results demonstrate that the spectral and basic copper binding properties of both methanobactins are similar and the unique copper binding capacity of both methanobactins lies primarily in the pair of five-membered rings and associated enethiol groups. The remaining portions of the methanobactin appear to provide the scaffolding that brings together of the two ring systems to produce the tetrahedral binding site for copper binding.

Hydroxylamine reduction to ammonium by plant and cyanobacterial hemoglobins.

Plants often face hypoxic stress as a result of flooding and waterlogged soils. During these periods, they must continue ATP production and nitrogen metabolism if they are to survive. The normal pathway of reductive nitrogen assimilation in non-legumes, nitrate, and nitrite reductase can be inhibited during low oxygen conditions that are associated with the buildup of toxic metabolites such as nitrite and nitric oxide, so the plant must also have a means of detoxifying these molecules. Compared to animal hemoglobins, plant and cyanobacterial hemoglobins are adept at reducing nitrite to nitric oxide under anaerobic conditions. Here we test their abilities to reduce hydroxylamine, a proposed intermediate of nitrite reductase, under anaerobic conditions. We find that class 1 rice nonsymbiotic hemoglobin (rice nsHb1) and the hemoglobin from the cyanobacterium Synechocystis (SynHb) catalyze the reduction of hydroxylamine to ammonium at rates 100-2500 times faster than animal hemoglobins including myoglobin, neuroglobin, cytoglobin, and blood cell hemoglobin. These results support the hypothesis that plant and cyanobacterial hemoglobins contribute to anaerobic nitrogen metabolism in support of anaerobic respiration and survival during hypoxia.

Plant and cyanobacterial hemoglobins reduce nitrite to nitric oxide under anoxic conditions.

The ability of ferrous hemoglobins to reduce nitrite to form nitric oxide has been demonstrated for hemoglobins from animals, including myoglobin, blood cell hemoglobin, neuroglobin, and cytoglobin. In all cases, the rate constants for the bimolecular reactions with nitrite are relatively slow, with maximal values of ~5 M(-1) s(-1) at pH 7. Combined with the relatively low concentrations of nitrite found in animal blood plasma (normally no greater than 13 µM), these slow reaction rates are unlikely to contribute significantly to hemoglobin oxidation, nitrite reduction, or NO production. Plants and cyanobacteria, however, must contend with much higher (millimolar) nitrite concentrations necessitated by assimilatory nitrogen metabolism during hypoxic growth, such as the conditions commonly found during flooding or in waterlogged soil. Here we report rate constants for nitrite reduction by a ferrous plant hemoglobin (rice nonsymbiotic hemoglobin 1) and a ferrous cyanobacterial hemoglobin from Synechocystis that are more than 10 times faster than those observed for animal hemoglobins. These rate constants, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant and cyanobacterial hemoglobins could serve as anaerobic nitrite reductases in vivo.

Crystal structures of Parasponia and Trema hemoglobins: differential heme coordination is linked to quaternary structure.

Hemoglobins from the plants Parasponia andersonii (ParaHb) and Trema tomentosa (TremaHb) are 93% identical in primary structure but differ in oxygen binding constants in accordance with their distinct physiological functions. Additionally, these proteins are dimeric, and ParaHb exhibits the unusual property of having different heme redox potentials for each subunit. To investigate how these hemoglobins could differ in function despite their shared sequence identity and to determine the cause of subunit heterogeneity in ParaHb, we have measured their crystal structures in the ferric oxidation state. Furthermore, we have made a monomeric ParaHb mutant protein (I43N) and measured its ferrous/ferric heme redox potential to test the hypothesized link between quaternary structure and heme heterogeneity in wild-type ParaHb. Our results demonstrate that TremaHb is a symmetric dimeric hemoglobin similar to other class 1 nonsymbiotic plant hemoglobins but that ParaHb has structurally distinct heme coordination in each of its two subunits that is absent in the monomeric I43N mutant protein. A mechanism for achieving structural heterogeneity in ParaHb in which the Ile(101(F4)) side chain contacts the proximal His(105(F8)) in one subunit but not the other is proposed. These results are discussed in the context of the evolution of plant oxygen transport hemoglobins, and other potential functions of plant hemoglobins.

Comparison of the dielectric response obtained from fluorescence upconversion measurements and molecular dynamics simulations for coumarin 153-apomyoglobin complexes and structural analysis of the complexes by NMR and fluorescence methods.

We present a comparison of the dielectric response obtained from fluorescence upconversion experiments and from molecular dynamics simulations of the complexes of coumarin 153 with five apomyoglobins (apoMbs): wild-type horse heart (HH-WT) and those of wild-type sperm whale (SW-WT); its two triple mutants, L29F/H64Q/V68F and H64L/V68F/P88A; and its double mutant, L29F/V68L. Comparisons between experimental and simulated solvation relaxation functions, C(t)s, for the wild-type proteins range from very good to excellent. For the three mutants we investigated, however, agreement between experiment and simulation was considerably inferior. Thus, an NMR study of the complex of the HH-WT complex apoMb, and fluorescence energy transfer and anisotropy studies of the five complexes, were performed to investigate the structures upon which the simulations were based. The NMR measurements confirm our earlier conclusions that the C153 lies in the heme pocket of the HH-WT apoMb. For the wild-type complexes, fluorescence energy transfer measurements provide two rise times, suggesting a definite spatial relationship between the two Trp donors and the C153 acceptor. These results confirm the structural integrity of the wild-type complexes and validate the initial structures used for the molecular dynamics simulations. On the other hand, the three mutants provided single exponential rise times for energy transfer, suggesting that the position of the C153 used in the simulations may have been in error or that the C153 is mobile on the time scale of the energy transfer experiment. Fluorescence anisotropy studies also suggest that the double mutant was not structurally intact. Furthermore, examination of these systems demonstrates the sensitivity of C153 to its environment and permits the observation of differences in the heme pockets. These results point to the importance of structural characterization of modified proteins used in studies of the dielectric response and suggest strategies for performing molecular dynamics simulations of modified proteins.

Structure and reactivity of hexacoordinate hemoglobins.

The heme prosthetic group in hemoglobins is most often attached to the globin through coordination of either one or two histidine side chains. Those proteins with one histidine coordinating the heme iron are called "pentacoordinate" hemoglobins, a group represented by red blood cell hemoglobin and most other oxygen transporters. Those with two histidines are called "hexacoordinate hemoglobins", which have broad representation among eukaryotes. Coordination of the second histidine in hexacoordinate Hbs is reversible, allowing for binding of exogenous ligands like oxygen, carbon monoxide, and nitric oxide. Research over the past several years has produced a fairly detailed picture of the structure and biochemistry of hexacoordinate hemoglobins from several species including neuroglobin and cytoglobin in animals, and the nonsymbiotic hemoglobins in plants. However, a clear understanding of the physiological functions of these proteins remains an elusive goal.

Trema and parasponia hemoglobins reveal convergent evolution of oxygen transport in plants.

All plants contain hemoglobins that fall into distinct phylogenetic classes. The subset of plants that carry out symbiotic nitrogen fixation expresses hemoglobins that scavenge and transport oxygen to bacterial symbiotes within root nodules. These "symbiotic" oxygen transport hemoglobins are distinct in structure and function from the nonoxygen transport ("nonsymbiotic") Hbs found in all plants. Hemoglobins found in two closely related plants present a paradox concerning hemoglobin structure and function. Parasponia andersonii is a nitrogen-fixing plant that expresses a symbiotic hemoglobin (ParaHb) characteristic of oxygen transport hemoglobins in having a pentacoordinate ferrous heme iron, moderate oxygen affinity, and a relatively rapid oxygen dissociation rate constant. A close relative that does not fix nitrogen, Trema tomentosa, expresses hemoglobin (TremaHb) sharing 93% amino acid identity to ParaHb, but its phylogeny predicts a typical nonsymbiotic hemoglobin with a hexacoordinate heme iron, high oxygen affinity, and slow oxygen dissociation rate constant. Here we characterize heme coordination and oxygen binding in TremaHb and ParaHb to investigate whether or not two hemoglobins with such high sequence similarity are actually so different in functional behavior. Our results indicate that the two proteins resemble nonsymbiotic hemoglobins in the ferric oxidation state and symbiotic hemoglobins in the ferrous oxidation state. They differ from each other only in oxygen affinity and oxygen dissociation rate constants, two factors key to their different functions. These results demonstrate distinct mechanisms for convergent evolution of oxygen transport in different phylogenetic classes of plant hemoglobins.

Review: correlations between oxygen affinity and sequence classifications of plant hemoglobins.

Plants express three phylogenetic classes of hemoglobins (Hb) based on sequence analyses. Class 1 and 2 Hbs are full-length globins with the classical eight helix Mb-like fold, whereas Class 3 plant Hbs resemble the truncated globins found in bacteria. With the exception of the specialized leghemoglobins, the physiological functions of these plant hemoglobins remain unknown. We have reviewed and, in some cases, measured new oxygen binding properties of a large number of Class 1 and 2 plant nonsymbiotic Hbs and leghemoglobins. We found that sequence classification correlates with distinct extents of hexacoordination with the distal histidine and markedly different overall oxygen affinities and association and dissociation rate constants. These results suggest strong selective pressure for the evolution of distinct physiological functions. The leghemoglobins evolved from the Class 2 globins and show no hexacoordination, very high rates of O(2) binding ( approximately 250 muM(-1) s(-1)), moderately high rates of O(2) dissociation ( approximately 5-15 s(-1)), and high oxygen affinity (K(d) or P(50) approximately 50 nM). These properties both facilitate O(2) diffusion to respiring N(2) fixing bacteria and reduce O(2) tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (K(HisE7) approximately 2), moderate rates of O(2) binding ( approximately 25 muM(-1) s(-1)), very small rates of O(2) dissociation ( approximately 0.16 s(-1)), and remarkably high O(2) affinities (P(50) approximately 2 nM), suggesting a function involving O(2) and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (K(HisE7) approximately 100), low rates of O(2) binding ( approximately 1 muM(-1) s(-1)), moderately low O(2) dissociation rate constants ( approximately 1 s(-1)), and moderate, Mb-like O(2) affinities (P(50) approximately 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.