Tyr(b10) Prevents Stabilization of Bound Oxygen in Soybean Leghemoglobin

Root nodules are complex structures that occur on roots of many agronomically important plants such as soybeans. Root nodules are a symbiosis between the plant and soil-borne bacteria. Once formed, and functional, root nodules can fix atmospheric nitrogen and provide the plant with a pollution-free source of nitrogen for seed production. A basic component of functional and efficient root nodules are the heme proteins called leghemoglobins. Leghemoglobins can bind to, and transport molecular oxygen and thereby maintain a low oxygen environment within the root nodule, permitting the microaerobic bacteria to convert atmospheric dinitrogen into ammonia, which is then assimilated by the plant for growth. Thus understanding the oxygen-binding characteristics of leghemoglobin are of major importance from both a basic and applied point of view. In this paper we have shown that specific amino acid residues are critical for allowing the orderly binding and transfer of oxygen from leghemoglobins. Although several hypotheses have been advanced to account for the biochemical properties of leghemoglobins, this work is the first that clearly demonstrates the molecular consequences of amino acids that surround the heme-pocket. Using engineered mutant proteins and spectroscopy, the role of Tyr (B10) has been investigated in detail for several vertebrate and plant hemoglobins

Effects of Distal Pocket Mutations on the Geminate Recombination of NO with Leghemoglobin on the Picosecond Time Scale

The picosecond NO geminate rebinding kinetics of wild-type leghemoglobin, a monomeric plant hemoglobin with structural similarity to myoglobin, and six mutant proteins at the distal histidine (H61G, H61A, H61V, H61L, H61R, H61F) are investigated. All of the mutant proteins yield rebinding kinetics that are initially more rapid than that of the wild-type protein. At long times, the rebinding of H61F becomes slower than that of wild-type leghemoglobin. The H61V, H61L, and H61G mutant proteins give extraordinarily rapid and complete geminate rebinding. On a 40 ps time scale, distal effects are overwhelmingly evident for all of the mutants considered. That binding is both rapid and, in several cases, essentially single-exponential is suggestive of the nature of the barrier induced by the distal modification:  it must be such that the ligand is prohibited from reorienting with respect to, and diffusing sufficiently far from, the heme iron so that a distribution of return paths is not offered to it. Over the past 20 years, the relative importance attributed to the proximal and the distal sides in modulating geminate ligand binding has varied considerably. Our results with leghemoglobin are discussed in terms of the relative contributions of proximal and distal effects to geminate rebinding kinetics

A Comparative Femtosecond Coherence Study of the Unligated Monomeric Hemeproteins Myoglobin and Leghemoglobin

Impulsive optical excitation has been performed on wild type, unligated leghemoglobin for the first time to compare the induced vibrational coherence with that observed in myoglobin. Both proteins were excited at the Soret maxima and probed at red and blue edges of the Soret band. The resulting kinetic traces were modulated by low-frequency vibrations. Leghemoglobin shows a decrease in vibrational amplitude compared with myoglobin. The possible cause for the amplitude differences is discussed in terms of contributions from both ground- and excited-state vibrational coherences and ground-state heterogeneity

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 O2 conditions. Results: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 Å 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 b5. 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 O2 reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O2 affinity is ultra-high (KD ≈ 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

Assessing the authority of political office-holders: the leadership capital index

This article argues that the extent to which political office-holders can effectively attain and wield authority is a function of the stock of ‘leadership capital.’ Drawing on the concept of political capital, we define leadership capital as aggregate authority composed of three dimensions: skills; relations; and reputation of a leader. Leadership capital ebbs and flows over time within a trajectory of acquisition, expenditure and inevitable depreciation. We present a Leadership Capital Index (LCI) that systematically maps out the three broad areas combining concrete measures with interpretive aspects. This can be used as a tool for systematically tracking and comparing the political fortunes of leaders in a way that is both more nuanced and robust than exclusive reliance on the latest approval ratings. We offer an illustrative case study of Tony Blair demonstrating the LCI. We conclude by discerning several promising paths for future development of the LCI

Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R g ~1.48-1.51 nm, helicity ~75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0-3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2-11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing.

Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4-14 laboratories. Six non-target viruses were detected by three or more laboratories. The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories