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.

Patterns of distribution of oxygen-binding globins, neuroglobin and cytoglobin in human retina.

OBJECTIVE: To determine the distribution of 2 intracellular oxygen-carrying molecules, neuroglobin (NGB) and cytoglobin (CYGB), in specific retinal cell types of human retinas. METHODS: Specific antibodies against NGB and CYGB were used in immunohistochemical studies to examine their distribution patterns in human retinal sections. Double-labeling studies were performed with the anti-NGB and anti-CYGB antibodies along with antibodies against neuronal (microtubule-associated protein 2, class III beta-tubulin [TUJ1], protein kinase C alpha, calretinin) and glial (vimentin, glial fibrillary acid protein) markers. Confocal microscopy was used to examine the retinal sections. RESULTS: Immunohistochemical analysis of human retinal tissue showed NGB and CYGB immunoreactivity in the ganglion cell layer, inner nuclear layer, inner and outer plexiform layers, and retinal pigment epithelium. Neuroglobin immunoreactivity was also present in the outer nuclear layer and photoreceptor inner segments. Neuroglobin and CYGB were coexpressed in the neurons in the ganglion cell layer and inner nuclear layer but not within glial cells. CONCLUSION: Neuroglobin and CYGB are colocalized within human retinal neurons and retinal pigment epithelium but not within glial cells. Clinical Relevance Our results suggest that NGB and CYGB may serve a neuroprotective role as scavengers of reactive oxygen species and therefore should be considered when developing therapeutic strategies for treatment of hypoxia-related ocular diseases.

Neuroglobin and cytoglobin distribution in the anterior eye segment: a comparative immunohistochemical study.

This study provides a detailed description of immunolocalization of two oxygen-binding proteins, neuroglobin (Ngb) and cytoglobin (Cygb), in the anterior segment of healthy human and canine eyes. Specific antibodies against Ngb and Cygb were used to examine their distribution patterns in anterior segment structures including the cornea, iris, trabecular meshwork, canal of Schlemm, ciliary body, and lens. Patterns of immunoreactivity (IR) were imaged with confocal scanning laser and conventional microscopy. Analysis of sectioned human and canine eyes showed Ngb and Cygb IR in the corneal epithelium and endothelium. In the iris, Ngb and Cygb IR was localized to the anterior border and the stroma, iridal sphincter, and dilator muscle. In the iridocorneal angle, Ngb and Cygb were detected in endothelial cells of the trabecular meshwork and canal of Schlemm in human. In the ciliary body, Ngb and Cygb IR was localized to the non-pigmented ciliary epithelium of the pars plana and pars plicata and in ciliary body musculature. Ngb and Cygb distribution was similar and colocalized within the same structures of healthy human and canine anterior eye segments. Based on their immunolocalization and previously reported biochemical features, we hypothesize that Ngb and Cygb may function as scavengers of reactive oxygen species. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

NO dioxygenase activity in hemoglobins is ubiquitous in vitro, but limited by reduction in vivo.

Genomics has produced hundreds of new hemoglobin sequences with examples in nearly every living organism. Structural and biochemical characterizations of many recombinant proteins reveal reactions, like oxygen binding and NO dioxygenation, that appear general to the hemoglobin superfamily regardless of whether they are related to physiological function. Despite considerable attention to "hexacoordinate" hemoglobins, which are found in nearly every plant and animal, no clear physiological role(s) has been assigned to them in any species. One popular and relevant hypothesis for their function is protection against NO. Here we have tested a comprehensive representation of hexacoordinate hemoglobins from plants (rice hemoglobin), animals (neuroglobin and cytoglobin), and bacteria (Synechocystis hemoglobin) for their abilities to scavenge NO compared to myoglobin. Our experiments include in vitro comparisons of NO dioxygenation, ferric NO binding, NO-induced reduction, NO scavenging with an artificial reduction system, and the ability to substitute for a known NO scavenger (flavohemoglobin) in E. coli. We conclude that none of these tests reveal any distinguishing predisposition toward a role in NO scavenging for the hxHbs, but that any hemoglobin could likely serve this role in the presence of a mechanism for heme iron re-reduction. Hence, future research to test the role of Hbs in NO scavenging would benefit more from the identification of cognate reductases than from in vitro analysis of NO and O(2) binding.

Plant hemoglobins: a molecular fossil record for the evolution of oxygen transport.

The evolution of oxygen transport hemoglobins occurred on at least two independent occasions. The earliest event led to myoglobin and red blood cell hemoglobin in animals. In plants, oxygen transport "leghemoglobins" evolved much more recently. In both events, pentacoordinate heme sites capable of inert oxygen transfer evolved from hexacoordinate hemoglobins that have unrelated functions. High sequence homology between hexacoordinate and pentacoordinate hemoglobins in plants has poised them for potential structural analysis leading to a molecular understanding of this important evolutionary event. However, the lack of a plant hexacoordinate hemoglobin structure in the exogenously ligand-bound form has prevented such comparison. Here we report the crystal structure of the cyanide-bound hexacoordinate hemoglobin from barley. This presents the first opportunity to examine conformational changes in plant hexacoordinate hemoglobins upon exogenous ligand binding, and reveals structural mechanisms for stabilizing the high-energy pentacoordinate heme conformation critical to the evolution of reversible oxygen binding hemoglobins.

Influence of the protein matrix on intramolecular histidine ligation in ferric and ferrous hexacoordinate hemoglobins.

Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly effected by the coordination and noncovalent chemical environment imposed by its connate protein. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here we explore this question by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria, including human neuroglobin and cytoglobin, a nonsymbiotic hemoglobin from rice, and a truncated hemoglobin from the cyanobacterium Synechocystis. This is done with a set of experiments measuring the reduction potentials of each wild-type hxHb and its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a noncoordinating side chain. These reduction potentials, coupled with studies of the mutant proteins saturated with exogenous imidazole, enable us to assess the effects of the protein matrices on histidine coordination. Our results show significant variation among the hxHbs, demonstrating flexibility in the globin moiety's ability to regulate reversible coordination. This regulation is particularly evident in the plant nonsymbiotic hemoglobins, where ferric state histidine coordination affinity is substantially lowered by the protein matrix.

Neuroglobin and cytoglobin: oxygen-binding proteins in retinal neurons.

PURPOSE: The goal of this study was to describe the detailed localization of the novel oxygen-binding molecules, neuroglobin (Ngb) and cytoglobin (Cygb), in mammalian retinas and to determine whether Ngb and Cygb are neuronal or glial proteins in the retina. METHODS: Antibodies directed against Ngb and Cygb were used to examine their patterns of distribution in normal canine retinas. Immunoblot analysis was performed to verify antibody specificity and the presence of Ngb and Cygb in canine tissues. Double-labeling immunohistochemistry was performed with the Ngb and Cygb antibodies along with antibodies against neuronal (MAP-2, class III beta-tubulin (TUJ1), PKCalpha, and calretinin) and glial antigens (vimentin and CRALBP). Tissue sections were analyzed with light and confocal microscopy. RESULTS: Ngb and Cygb proteins were observed in different retinal cells. Cygb (but not Ngb) was also present in canine kidney, liver, lung, and heart tissue. Immunohistochemical analysis of canine retinas demonstrated Ngb immunoreactivity (IR) in the ganglion cell layer (GCL), inner (INL) and outer (ONL) nuclear layers, inner (IPL) and outer plexiform (OPL) layers, photoreceptor inner segments (IS), and retinal pigment epithelium (RPE). Ngb IR was localized within retinal neurons, but not in glia. Cygb IR was found in neurons and their processes in the GCL, IPL, INL, and OPL and within the RPE, but not in glia. CONCLUSIONS: Ngb and Cygb are widely distributed in retinal neurons and RPE, but not in glial cells of the canine retina. Their structure and distribution is suggestive of a possible role in oxygen transport in the mammalian retina.

Bis-histidyl hexacoordination in hemoglobins facilitates heme reduction kinetics.

Hexacoordinate hemoglobins are a class of proteins that exhibit reversible bis-histidyl coordination of the heme iron while retaining the ability to bind exogenous ligands. One hypothesis for their physiological function is that they scavenge nitric oxide, a reaction that oxidizes the protein and requires reduction of the heme iron to continue. Reduction kinetics of hexacoordinate hemoglobins, including human neuroglobin and cytoglobin, and those from Synechocystis and rice, are compared to myoglobin, soybean leghemoglobin, and several relevant mutant proteins. In all cases, bis-histidyl coordination greatly increases the rate of reduction by sodium dithionite when compared to pentacoordinate hemoglobins. In myoglobin and leghemoglobin, reduction is limited by the rate constant for electron transfer, whereas in the hexacoordinate hemoglobins reduction is limited only by bimolecular binding of the reductant. These results can be explained by differences in the reorganization energy for reduction between hexacoordinate and pentacoordinate hemoglobins.

Crystallographic analysis of synechocystis cyanoglobin reveals the structural changes accompanying ligand binding in a hexacoordinate hemoglobin.

The crystal structures of cyanide and azide-bound forms of the truncated hemoglobin from Synechocystis are presented at 1.8 angstroms resolution. A comparison with the structure of the endogenously liganded protein reveals a conformational shift unprecedented in hemoglobins, and provides the first picture of a hexacoordinate hemoglobin in both the bis-histidyl and the exogenously coordinated states. The structural changes between the different conformations are confined to two regions of the protein; the B helix, and the E helix, including the EF loop. A molecular "hinge" controlling movement of the E helix is observed in the EF loop, which is composed of three principal structural elements: Arg64, the heme-d-propionate, and a three-residue extension of the F helix. Additional features of the structural transition between the two protein conformations are discussed as they relate to the complex ligand-binding behavior observed in hexacoordinate hemoglobins, and the potential physiological function of this class of proteins.

The crystal structure of Synechocystis hemoglobin with a covalent heme linkage.

The x-ray crystal structure of Synechocystis hemoglobin has been solved to a resolution of 1.8 A. The conformation of this structure is surprisingly different from that of the previously reported solution structure, probably due in part to a covalent linkage between the heme 2-vinyl and His117 that is present in the crystal structure but not in the structure solved by NMR. Synechocystis hemoglobin is a hexacoordinate hemoglobin in which the heme iron is coordinated by both the proximal and distal histidines. It is also a member of the "truncated hemoglobin" family that is much shorter in primary structure than vertebrate and plant hemoglobins. In contrast to other truncated hemoglobins, the crystal structure of Synechocystis hemoglobin displays no "ligand tunnel" and shows that several important amino acid side chains extrude into the solvent instead of residing inside the heme pocket. The stereochemistry of hexacoordination is compared with other hexacoordinate hemoglobins and cytochromes in an effort to illuminate factors contributing to ligand affinity in hexacoordinate hemoglobins.

Plants, humans and hemoglobins.

New developments have forced a re-evaluation of our understanding of the structure and function of hemoglobins. Leghemoglobins regulate oxygen affinity through a mechanism different from that of myoglobin using a novel combination of heme pocket amino acids that lower the oxygen affinity. The hexacoordinate hemoglobins are characterized by intramolecular coordination of the ligand binding site at the heme iron, and were first identified in plants as the 'non-symbiotic plant hemoglobins'. They are now known to be present in animals and bacteria. Many of these proteins are upregulated in both plants and animals during hypoxia or similar stresses. Therefore, there might be a common physiological function for hexacoordinate hemoglobins in plants and animals.

Direct measurement of equilibrium constants for high-affinity hemoglobins.

The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) << 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.

A ubiquitously expressed human hexacoordinate hemoglobin.

We have identified a new human hemoglobin that we call histoglobin because it is expressed in a wide array of tissues. Histoglobin shares less than 30% identity with the other human hemoglobins, and the gene contains an intron in an unprecedented location. Spectroscopic and kinetic experiments with recombinant human histoglobin indicate that it is a hexacoordinate hemoglobin with significantly different ligand binding characteristics than the other human hexacoordinate hemoglobin, neuroglobin. In contrast to the very high oxygen affinities displayed by most hexacoordinate hemoglobins, the biophysical characteristics of histoglobin indicate that it could facilitate oxygen transport. The discovery of histoglobin demonstrates that humans, like plants, differentially express multiple hexacoordinate hemoglobins.