Structural characterization of a group II 2/2 hemoglobin from the plant pathogen Agrobacterium tumefaciens.

Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens (At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1Å resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

On optima: the case of myoglobin-facilitated oxygen diffusion.

The process of myoglobin/leghemoglobin-facilitated oxygen diffusion is adapted to function in different environments in diverse organisms. We enquire how the functional parameters of the process are optimized in particular organisms. The ligand-binding properties of the proteins, myoglobin and plant symbiotic hemoglobins, we discover, suggest that they have been adapted under genetic selection pressure for optimal performance. Since carrier-mediated oxygen transport has probably evolved independantly many times, adaptation of diverse proteins for a common functionality exemplifies the process of convergent evolution. The progenitor proteins may be built on the myoglobin scaffold or may be very different.

Myoglobin-enhanced oxygen delivery to isolated cardiac mitochondria.

The heart, red skeletal muscles and the nitrogen-fixing legume root nodule function in steady states of high oxygen influx, partial oxygenation of cytoplasmic myoglobin or leghemoglobin and correspondingly low oxygen partial pressure. Here, we ask: what conditions are required at the surface of actively respiring, state III, tightly coupled mitochondria to enhance oxygen flow to cytochrome oxidase? Pigeon heart mitochondria were isolated with minimal damage to the outer mitochondrial membrane and were incubated at low oxygen pressures, where respiration is oxygen limited, with solutions of each of six monomeric hemoglobins with widely divergent kinetics and equilibria in their reactions with oxygen: Busycon myoglobin, horse myoglobin, Lucina hemoglobins I and II, soybean leghemoglobin c and Gasterophilus hemoglobin. Each augments oxygen uptake. The declining fractional saturation of each hemoglobin with oxygen was monitored spectrophotometrically as mitochondrial respiration depleted the oxygen; the oxygen partial pressure at half-maximal rate of oxygen uptake was similar for each hemoglobin, supporting the conclusion that the hemoglobins did not interact with the mitochondrial surface in oxygen delivery. The oxygen pressure required to support state III mitochondrial oxygen uptake, 0.005 kPa (0.04 torr), is small compared with that obtained in the sarcoplasm and at the mitochondrial surface of the working heart, 0.32 kPa (2.4 torr). We conclude that, in normal steady states of contraction of the myoglobin-containing heart, oxygen utilization by mitochondrial cytochrome oxidase is not limited by oxygen availability.

Reaction of Mycobacterium tuberculosis truncated hemoglobin O with hydrogen peroxide: evidence for peroxidatic activity and formation of protein-based radicals.

In this work, we investigated the reaction of ferric Mycobacterium tuberculosis truncated hemoglobin O (trHbO) with hydrogen peroxide. Stopped-flow spectrophotometric experiments under single turnover conditions showed that trHbO reacts with H(2)O(2) to give transient intermediate(s), among which is an oxyferryl heme, different from a typical peroxidase Compound I (oxyferryl heme pi-cation radical). EPR spectroscopy indicated evidence for both tryptophanyl and tyrosyl radicals, whereas redox titrations demonstrated that the peroxide-treated protein product retains 2 oxidizing eq. We propose that Compound I formed transiently is reduced with concomitant oxidation of Trp(G8) to give the detected oxoferryl heme and a radical on Trp(G8) (detected by EPR of the trHbO Tyr(CD1)Phe mutant). In the wild-type protein, the Trp(G8) radical is in turn reduced rapidly by Tyr(CD1). In a second cycle, Trp(G8) may be reoxidized by the ferryl heme to yield ferric heme and two protein radicals. In turn, these migrate to form tyrosyl radicals on Tyr(55) and Tyr(115), which lead, in the absence of a reducing substrate, to oligomerization of the protein. Steady-state kinetics in the presence of H(2)O(2) and the one-electron donor 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) indicated that trHbO has peroxidase activity, in accord with the presence of typical peroxidase intermediates. These findings suggest an oxidation/reduction function for trHbO and, by analogy, for other Group II trHbs.

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins.

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.

Viscosity-dependent relaxation significantly modulates the kinetics of CO recombination in the truncated hemoglobin TrHbN from Mycobacterium tuberculosis.

Kinetic traces were generated for the nanosecond and slower rebinding of photodissociated CO to trHbN in solution and in porous sol-gel matrices as a function of viscosity, conformation, and mutation. TrHbN is one of the two truncated hemoglobins from Mycobacterium tuberculosis. The kinetic traces were analyzed in terms of three distinct phases. These three phases are ascribed to rebinding: (i) from the distal heme pocket, (ii) from the adjacent apolar tunnel prior to conformational relaxation, and (iii) from the apolar tunnel subsequent to conformational relaxation. The fractional content of each of these phases was shown to be a function of the viscosity and, in the case of the sol-gel-encapsulated samples, sample preparation history. The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand. Implications of these finding with respect to biological function are discussed.

Reactions of Mycobacterium tuberculosis truncated hemoglobin O with ligands reveal a novel ligand-inclusive hydrogen bond network.

Truncated hemoglobin O (trHbO) is one of two trHbs in Mycobacterium tuberculosis. Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may be important in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate the reactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhanced resonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociation of O(2) and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0 microM(-1) s(-1)) and dissociation (<0.006 s(-1)) of O(2) are among the slowest known, precluding transport or diffusion of O(2) as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this group controls ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants for O(2) and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicates that the chi((2,1)) dihedral angle for the indole ring increases progressively from approximately 93 degrees to at least 100 degrees in going sequentially from the deoxy to CO to O(2) derivative, demonstrating a significant conformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a network of hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues being within hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding network may thus represent a considerable barrier to ligand entrance and escape. In accord with this model, we found that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O(2) dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligand escape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivative of myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by the hydrogen bonding network.

Myoglobin function reassessed.

The heart and those striated muscles that contract for long periods, having available almost limitless oxygen, operate in sustained steady states of low sarcoplasmic oxygen pressure that resist change in response to changing muscle work or oxygen supply. Most of the oxygen pressure drop from the erythrocyte to the mitochondrion occurs across the capillary wall. Within the sarcoplasm, myoglobin, a mobile carrier of oxygen, is developed in response to mitochondrial demand and augments the flow of oxygen to the mitochondria. Myoglobin-facilitated oxygen diffusion, perhaps by virtue of reduction of dimensionality of diffusion from three dimensions towards two dimensions in the narrow spaces available between mitochondria, is rapid relative to other parameters of cell respiration. Consequently, intracellular gradients of oxygen pressure are shallow, and sarcoplasmic oxygen pressure is nearly the same everywhere. Sarcoplasmic oxygen pressure, buffered near 0.33 kPa (2.5 torr; equivalent to approximately 4 micro mol l(-1) oxygen) by equilibrium with myoglobin, falls close to the operational K(m) of cytochrome oxidase for oxygen, and any small increment in sarcoplasmic oxygen pressure will be countered by increased oxygen utilization. The concentration of nitric oxide within the myocyte results from a balance of endogenous synthesis and removal by oxymyoglobin-catalyzed dioxygenation to the innocuous nitrate. Oxymyoglobin, by controlling sarcoplasmic nitric oxide concentration, helps assure the steady state in which inflow of oxygen into the myocyte equals the rate of oxygen consumption.

Kinetic modulation in carbonmonoxy derivatives of truncated hemoglobins: the role of distal heme pocket residues and extended apolar tunnel.

Truncated hemoglobins (trHbs), are a distinct and newly characterized class of small myoglobin-like proteins that are widely distributed in bacteria, unicellular eukaryotes, and higher plants. Notable and distinctive features associated with trHbs include a hydrogen-bonding network within the distal heme pocket and a long apolar tunnel linking the external solvent to the distal heme pocket. The present work compares the geminate and solvent phase rebinding kinetics from two trHbs, one from the ciliated protozoan Paramecium caudatum (P-trHb) and the other from the green alga Chlamydomonas eugametos (C-trHb). Unusual kinetic patterns are observed including indications of ultrafast (picosecond) geminate rebinding of CO to C-trHb, very fast solvent phase rebinding of CO for both trHbs, time-dependent biphasic CO rebinding kinetics for P-trHb at low CO partial pressures, and for P-trHb, an increase in the geminate yield from a few percent to nearly 100% under high viscosity conditions. Species-specific differences in both the 8-ns photodissociation quantum yield and the rebinding kinetics, point to a pivotal functional role for the E11 residue. The response of the rebinding kinetics to temperature, ligand concentration, and viscosity (glycerol, trehalose) and the viscosity-dependent changes in the resonance Raman spectrum of the liganded photoproduct, together implicate both the apolar tunnel and the static and dynamic properties of the hydrogen-bonding network within the distal heme pocket in generating the unusual kinetic patterns observed for these trHbs.