Mark S. Hargrove

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.

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 O2 binding.

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 O2 binding (∼250 μM−1 s−1), moderately high rates of O2 dissociation (∼5–15 s−1), and high oxygen affinity (Kd or P50 ≈ 50 nM). These properties both facilitate O2 diffusion to respiring N2 fixing bacteria and reduce O2 tension in the root nodules of legumes. The Class 1 plant Hbs show weak hexacoordination (KHisE7 ≈ 2), moderate rates of O2 binding (∼25 μM−1 s−1), very small rates of O2 dissociation (∼0.16 s−1), and remarkably high O2 affinities (P50 ≈ 2 nM), suggesting a function involving O2 and nitric oxide (NO) scavenging. The Class 2 Hbs exhibit strong hexacoordination (KHisE7 ≈ 100), low rates of O2 binding (∼1 μM−1 s−1), moderately low O2 dissociation rate constants (∼1 s−1), and moderate, Mb‐like O2 affinities (P50 ≈ 340 nM), perhaps suggesting a sensing role for sustained low, micromolar levels of oxygen.

The structure and function of plant hemoglobins

Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin exist in plants: symbiotic, non-symbiotic, and truncated hemoglobins. Crystal structures and other structural and biophysical techniques have revealed important knowledge about ligand binding and conformational stabilization in all three types. In symbiotic hemoglobins (leghemoglobins), ligand binding regulatory mechanisms have been shown to differ dramatically from myoglobin and red blood cell hemoglobin. In the non-symbiotic hemoglobins found in all plants, crystal structures and vibrational spectroscopy have revealed the nature of the structural transition between the hexacoordinate and ligand-bound states. In truncated hemoglobins, the abbreviated globin is porous, providing tunnels that may assist in ligand binding, and the bound ligand is stabilized by more than one distal pocket residue. Research has implicated these plant hemoglobins in a number of possible functions differing among hemoglobin types, and possibly between plant species.

A flash photolysis method to characterize hexacoordinate hemoglobin kinetics.

A flash photolysis method is described for analyzing ligand binding to the new and growing group of hemoglobins which are hexacoordinate in the unligated, ferrous state. Simple analysis of a two exponential fit to time courses for CO rebinding at varying CO concentrations yields rate constants for formation and dissociation of the hexacoordinate complex, and the bimolecular rate constant for CO binding. This method was tested with a nonsymbiotic plant hemoglobin from rice for which these values had not previously been determined. For this protein, dissociation and rebinding of the hexacoordinating amino acid side chain, His(73), is rapid and similar to the rate of CO binding at high CO concentrations. These results indicate that hexacoordination must be taken into account when evaluating the affinity of hexacoordinate hemoglobins for ligands.

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.

Weedy adaptation in Setaria spp. V. Effects of gaseous environment on giant foxtail (Setaria faberii) (Poaceae) seed germination

The effects that naturally occurring gases (oxygen, nitrogen, carbon monoxide) may cause in dormant giant foxtail (Setaria faberii) seed germination under favorable temperature and moisture conditions were investigated. The germination responses to gas mixtures supported the hypothesis that S. faberii germination behavior is regulated by the amount of oxygen taken into hydrated seed over time. Setaria faberii seed germination was markedly affected by O(2) concentration (in N(2)) above and below that of air (20% O(2)): the largest increase in germination (from 37 to 60%) occurred between 20-25% O(2); between 0-10% O(2), germination increased from 0-30%; and surprisingly germination at 10 and 20% O(2) was similar. These observations reveal an asymmetrical response to incremental changes in O(2) above and below that typically found in agricultural soils. Carbon monoxide had opposite effects on S. faberii germination in air depending on concentration, stimulation, and inhibition: germination increased from 37 to 56% with the addition of 1% CO, but decreased from 37 to 14% with 75% added CO. An explanation may be that there are two separate effects of CO, each occurring in different physiological systems of dormant seeds at the same time. At high concentrations (75%) in air CO inhibited seed germination, probably by inhibiting mitochondrial respiration. But low CO concentrations (0.1 or 1%) in air stimulated seed germination. It was not apparent which physiological system(s) CO and O(2) affected. It seems unlikely that CO-stimulated germination arises from effects on the respiratory apparatus, but may be a consequence of CO interactions with an as yet unknown physiological factor in the seed. We provide a model of Setaria spp. dormancy consistent with its seed morphology, the gas-germination data, and the hypothesized second physiological factor that may be involved in CO stimulated germination.

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F‐helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F‐helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen‐bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function. Proteins 2002;46:268–277.

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 moietys 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. Proteins 2007.

Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin.

Leghemoglobins facilitate diffusion of oxygen through root tissue to a bacterial terminal oxidase in much the same way that myoglobin transports oxygen from blood to muscle cell mitochondria. Leghemoglobin serves an additional role as an oxygen scavenger to prevent inhibition of nitrogen fixation. For this purpose, the oxygen affinity of soybean leghemoglobin is 20‐fold greater than myoglobin, resulting from an 8‐fold faster association rate constant combined with a 3‐fold slower dissociation rate constant. Although the biochemical mechanism used by myoglobin to bind oxygen has been described in elegant detail, an explanation for the difference in affinity between these two structurally similar proteins is not obvious. The present work demonstrates that, despite their similar structures, leghemoglobin uses methods different from myoglobin to regulate ligand affinity. Oxygen and carbon monoxide binding to a comprehensive set of leghemoglobin distal heme pocket mutant proteins in comparison to their myoglobin counterparts has revealed some of these mechanisms. The “distal histidine” provides a crucial hydrogen bond to stabilize oxygen in myoglobin but has little effect on bound oxygen in leghemoglobin and is retained mainly for reasons of protein stability and prevention of heme loss. Furthermore, soybean leghemoglobin uses an unusual combination of HisE7 and TyrB10 to sustain a weak stabilizing interaction with bound oxygen. Thus, the leghemoglobin distal heme pocket provides a much lower barrier to oxygen association than occurs in myoglobin and oxygen dissociation is regulated from the proximal heme pocket. Proteins 2003;50:239–248.