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.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.     

A circularly permuted myoglobin possesses a folded structure and ligand binding similar to those of the wild-type protein but with a reduced thermodynamic stability

A circular permutein of sperm whale myoglobin in which the G helix is C-terminal, the H helix is N-terminal, and 16 amino acids link the H helix to the A helix has been expressed in Escherichia coli. The permutein sequence begins with Gly121 (using the numbering scheme for the wild-type protein) and terminates with Pro120. The ligand binding function of the permutein was assayed using stopped-flow methods and shown to be essentially identical to that of the wild-type protein. In addition, one- and two-dimensional NMR studies of the cyanomet isoform of the permutein show a nativelike structure with a heme binding pocket very similar to that of the wild-type myoglobin. Although the structure and function of the permutein resemble those of the wild-type myoglobin, the permutein is less stable to chemical denaturation by 5.2 kcal/mol.     

A simplified mechanical model of proteins tested on the globin fold.

It has been shown that the distribution of presently known protein loop lengths is consistent with even the simplest available theory of rubber-like elasticity, and with the idea that such loops generate an entropically derived end-to-end tension. It has also been asserted that the molten globule phase, just like the native form, must be mechanically stable, and that a simple demonstration of the potential for mechanical stability would be a powerful test in predictions of new protein folds. This paper amplifies this suggestion by explicit calculation of a familiar but non-trivial test case: sperm-whale myoglobin. The method used is to describe the protein molecule in terms of a highly simplified mechanical model bearing some resemblance to a pre-stressed mechanism. The alpha-helices are treated as rigid rods and the loops are treated as elastic strings. The entropic tensions exerted by the loops are imposed on the mechanism using an approximation proposed earlier. The helices are then held to generate frictionless reaction forces at their mutual points of contact. These contact forces are calculated to null out maximally the effects of the loop tensions, and hence stabilize the molecule. It is shown that the crystallographically determined structure of myoglobin has a significantly higher mechanical stability on this model than does any of a previously published set of combinatorially generated predictions. Amongst the predictions alone, the best is also the one with the highest stability. It is anticipated that this result could be of general importance in sorting or filtering out bad predictions. A further exciting feature of the model is that it offers a natural explanation for the strong conservation of the C2 proline and the invariably long unconserved sequence from the end of the C helix to the start of the E helix in the globins and phycocyanins.     

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.     

Use of site-directed mutagenesis to destabilize native apomyoglobin relative to folding intermediates

Site-directed mutagenesis has been used to study the effect on the stability of human apomyoglobin (apoMb) of modifying the size, hydrophobicity, and charge of a central residue in the G.B helix-helix packing interface. Some stability measurements have also been made on the corresponding holomyoglobins (heme present). Cys-110, a central helix pairing residue in the G helix, has been changed to Ala, Ser, Asp, and Leu. Stability to low-pH-induced unfolding has been measured for both native apoMb and the compact folding intermediate discovered by Griko et al. [Griko, Y. V., Privalov, P. L., Venyaminov, S. Y., & Kutyshenko, V. P. (1988) J. Mol. Biol. 202, 127-138]. As judged by its circular dichroism spectrum, this intermediate has a substantial helix content (about 35%). Whether or not this inferred helical structure is closely related to the myoglobin structure is not yet known. The mutational evidence shows that integrity of G.B helix pairing is important for the stability of apoMb as well as of myoglobin and that this helix pairing site is very sensitive to both steric and electrostatic disruption. Our results also suggest that G.B helix pairing does not stabilize the compact intermediate; hence, disrupting this site destabilizes the native protein relative to the compact intermediate. Such selective destabilization of the native state relative to equilibrium folding intermediates is not restricted to acid denaturation: urea denaturation of the Leu mutant appears to display at least one stable intermediate, while wild-type and the remaining mutant apoMbs undergo two-state urea unfolding transitions.     

Variation of folded polypeptide surface area with probe size

Three types of polypeptide surface area (contact, accessible, and molecular) have been studied as a function of the radius of a probe sphere used to map the surface. The surfaces are: (1) three alpha-helices, the H-helix of myoglobin, the E-helix of leghemoglobin, and an artificial polyalanine helix, each with 26 residues; (2) two globins, myoglobin and leghemoglobin, each with 153 residues; and (3) a two-center model system for which the three types of surface area have been calculated analytically. The two globin helices have almost identical surface areas as a function of probe size as do the two globins. The polyalanine helix surface area is smaller but similar in shape to the globin helix areas. All three helix contact areas tend to the same limit as the probe size increases, and the globin contact areas behave similarly. Fractal dimensions were calculated for the helix and globin contact and molecular surfaces. All fractal dimensions showed strong dependence on probe size. The contact fractal dimension peaks at larger values for both the helices and globins. Most residues do not make contact with large probes (15 A).     

Functional effects of heme orientational disorder in sperm whale myoglobin

The optical absorption and ligand binding properties of newly reconstituted sperm whale myoglobin were examined systematically at pH 8, 20 degrees C. The conventional absorbance and magnetic circular dichroism spectra of freshly reconstituted samples were identical to those of the native protein. In contrast, reconstituted azide or CO myoglobin initially exhibited less circular dichroism in the Soret wavelength region than native myoglobin. These data support the theory proposed by La Mar and co-workers (La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, R. M. (1983) J. Mol. Biol. 168, 887-896) that protoheme inserts into apomyoglobin in two distinct orientations. The equilibrium and kinetic parameters for O2 and CO binding to newly reconstituted myoglobin were observed to be identical to those of the native protein. Thus, the orientation of the heme group has no effect on the physiological properties of myoglobin. This result is in disagreement with the preliminary report of Livingston et al. (Livingston, D. J., Davis, N. L., La Mar, G. N., and Brown, W. D. (1984) J. Am. Chem. Soc. 106, 3025-3026) which suggested that the abnormal heme conformation exhibited a 10-fold greater affinity and association rate constant for O2 binding. Significant kinetic heterogeneity was observed only for long-chain isonitrile binding to newly reconstituted myoglobin, and even in these cases, the rate constants for the abnormal and normal heme conformations differed by less than a factor of 4.     

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.     

Gamma-glutamyltransferase activity of liver plasma membrane: induction following chronic alcohol consumption

The proton nuclear magnetic resonance spectra of soybean ferric leghemoglobin $\text{a}$ in the low-spin cyanide and nicotinate complexes have been assigned by specific deuteration of heme methyl groups. The assignments differ from those obtained solely from nuclear Overhauser enhancement measurements and are indicative of a proximal histidyl imidazole-hemin interaction which is very similar to that found in sperm whale myoglobin. The absence of a hyperfine shifted exchangeable NH peak for the distal histidine in leghemoglobin suggests either a very different orientation for this distal ligand or a significantly faster exchange rate with bulk solvent than found in myoglobin.     

Kinetics and thermodynamics of oxygen, CO, and azide binding by the subcomponents of soybean leghemoglobin

Leghemoglobin shows extreme high affinity behavior in the binding of both oxygen and CO. We have determined the temperature dependence of the rate constants for ligation of oxygen and CO and from these data the thermodynamics (delta G0, delta H0, delta S0) of ligation for the purified components of soybean leghemoglobin. X-ray crystallography has shown that the heme cavity can easily accommodate ligands the size of nicotinate, and analysis of extended x-ray absorption fine structure data has shown that the Fe atom is in the mean plane of the heme in the leghemoglobin-CO complex. Ligation of oxygen and CO are in accord with this picture in that the Ea for oxygen binding is that expected for a diffusion controlled reaction and delta S0 for the ligation of both CO and oxygen is consistent with the simple immobilization of the ligand at the Fe, with no evidence for significant conformational changes in the protein or changes in solvation. At 20 degrees C the rate constants for oxygen and CO binding vary by 26-44% among the eight leghemoglobin components. For azide binding the variation is a factor of 2. These variations appear to arise from amino acid substitutions outside either the heme cavity or the two major paths for ligand entry to the heme. The distribution of leghemoglobin components varies with the age of the soybean nodule during the growing season. The changes in composition alone, however, would only allow the concentration of free oxygen to vary by about 3%. This finding calls into question models that ascribe a significant functional role to changes in the distribution of leghemoglobin components in regulating oxygen concentration in the nodule.     

NMR studies of the conformations of leghemoglobins from soybean and lupin

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.     

Hydrogen exchange behavior of [U-15N]-labeled oxidized and reduced iso-1-cytochrome c

Heteronuclear NMR spectroscopy was used to measure the hydrogen-deuterium exchange rates of backbone amide hydrogens in both oxidized and reduced [U-15N]iso-1-cytochrome c from the yeast Saccharomyces cerevisiae. The exchange data confirm previously reported data [Marmorino et al. (1993) Protein Sci. 2, 1966-1974], resolve several inconsistencies, and provide more thorough coverage of exchange rates throughout the cytochrome c protein in both oxidation states. Combining the data previously collected on unlabeled C102T with the current data collected on [U-15N]C102T, exchange rates for 53 protons in the oxidized state and 52 protons in the reduced state can now be reported. Most significantly, hydrogen exchange measurements on [U-15N]iso-1-cytochrome c allowed the observation of exchange behavior of the secondary structures, such as large loops, that are not extensively hydrogen-bonded. For the helices, the most slowly exchanging protons are found in the middle of the helix, with more rapidly exchanging protons at the helix ends. The observation for the Omega-loops in cytochrome c is just the opposite. In the loops, the ends contain the most slowly exchanging protons and the loop middles allow more rapid exchange. This is found to be true in cytochrome c loops, even though the loop ends are not attached to any regular secondary structures. Some of the exchange data are strikingly inconsistent with data collected on the C102S variant at a different pH, which suggests pH-dependent dynamic differences in the protein structure. This new hydrogen exchange data for loop residues could have implications for the substructure model of eukaryotic cytochrome c folding. Isotopic labeling of variant forms of cytochrome c can now be used to answer many questions about the structure and folding of this model protein.     

Identification of ligand effector binding sites in transmembrane regions of the human G protein-coupled C3a receptor

The human C3a anaphylatoxin receptor (C3aR) is a G protein-coupled receptor (GPCR) composed of seven transmembrane alpha-helices connected by hydrophilic loops. Previous studies of chimeric C3aR/C5aR and loop deletions in C3aR demonstrated that the large extracellular loop2 plays an important role in noneffector ligand binding; however, the effector binding site for C3a has not been identified. In this study, selected charged residues in the transmembrane regions of C3aR were replaced by Ala using site-directed mutagenesis, and mutant receptors were stably expressed in the RBL-2H3 cell line. Ligand binding studies demonstrated that R161A (helix IV), R340A (helix V), and D417A (helix VII) showed no binding activity, although full expression of these receptors was established by flow cytometric analysis. C3a induced very weak intracellular calcium flux in cells expressing these three mutant receptors. H81A (helix II) and K96A (helix III) showed decreased ligand binding activity. The calcium flux induced by C3a in H81A and K96A cells was also consistently reduced. These findings suggest that the charged transmembrane residues Arg161, Arg340, and Asp417 in C3aR are essential for ligand effector binding and/or signal coupling, and that residues His81 and Lys96 may contribute less directly to the overall free energy of ligand binding. These transmembrane residues in C3aR identify specific molecular contacts for ligand interactions that account for C3a-induced receptor activation.     

MacA is a second cytochrome c peroxidase of Geobacter sulfurreducens

The metal-reducing δ-proteobacterium Geobacter sulfurreducens produces a large number of c-type cytochromes, many of which have been implicated in the transfer of electrons to insoluble metal oxides. Among these, the dihemic MacA was assigned a central role. Here we have produced G. sulfurreducens MacA by recombinant expression in Escherichia coli and have solved its three-dimensional structure in three different oxidation states. Sequence comparisons group MacA into the family of diheme cytochrome c peroxidases, and the protein indeed showed hydrogen peroxide reductase activity with ABTS(-2) as an electron donor. The observed K(M) was 38.5 ± 3.7 μM H(2)O(2) and v(max) was 0.78 ± 0.03 μmol of H(2)O(2)·min(-1)·mg(-1), resulting in a turnover number k(cat) = 0.46 · s(-1). In contrast, no Fe(III) reductase activity was observed. MacA was found to display electrochemical properties similar to other bacterial diheme peroxidases, in addition to the ability to electrochemically mediate electron transfer to the soluble cytochrome PpcA. Differences in activity between CcpA and MacA can be rationalized with structural variations in one of the three loop regions, loop 2, that undergoes conformational changes during reductive activation of the enzyme. This loop is adjacent to the active site heme and forms an open loop structure rather than a more rigid helix as in CcpA. For the activation of the protein, the loop has to displace the distal ligand to the active site heme, H93, in loop 1. A H93G variant showed an unexpected formation of a helix in loop 2 and disorder in loop 1, while a M297H variant that altered the properties of the electron transfer heme abolished reductive activation.     

Comparison of simulated and experimentally determined dynamics for a variant of the Lacl DNA-binding domain, Nlac-P.

Recent advances in the experimentally determined structures and dynamics of the domains within LacI provide a rare context for evaluating dynamics calculations. A 1500-ps trajectory was simulated for a variant of the LacI DNA-binding domain, which consists of the first three helices in LacI and the hinge helix of the homologous PurR. Order parameters derived from dynamics simulations are compared to those obtained for the LacI DNA-binding domain with 15N relaxation NMR spectroscopy (Slijper et al., 1997. Biochemistry. 36:249-254). The MD simulations suggest that the unstructured loop between helices II and III does not exist in a discrete state under the conditions of no salt and neutral pH, but occupies a continuum of states between the DNA-bound and free structures. Simulations also indicate that the unstructured region between helix III and the hinge helix is very mobile, rendering motions of the hinge helix essentially independent of the rest of the protein. Finally, the alpha-helical hydrogen bonds in the hinge helix are broken after 1250 ps, perhaps as a prelude to helix unfolding.     

Contribution of protein conformation to heme stereochemistry and reactivity. Low-temperature magnetic circular dichroism data

Visible and near infrared magnetic circular dichroism (MCD) spectra of heme proteins and enzymes as well as those of a protein-free heme bound to 2-methylimidazole were recorded and compared at 4.2 K in unrelaxed metastable and relaxed equilibrium heme stereochemistry. The relaxed and unrelaxed stereochemistries of a 5-coordinate ferrous heme were generated by chemical reduction of iron at room temperature before freezing the sample and by photolysis of CO or O2 complexes at 4.2 K, respectively. The results are discussed in terms of a protein contribution into energies of the Fe-N epsilon(His) and Fe-N(pyrrols) bonds and their change on a ligand binding. We observed and analyzed cases of weak (myoglobin, hemoglobin) and strong (leghemoglobin, peroxidases) constraints imposed by the protein conformation on the proximal heme stereochemistry by comparing the bond energies in proteins with those in the protoheme-(2-methylimidazole) model compound. The role of a protein moiety in modulating the ligand binding properties of leghemoglobin and the heme reactivity of horseradish peroxidase is discussed.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.     

Circular permutation and deletion studies of myoglobin indicate that the correct position of its N-terminus is required for native stability and solubility but not for native-like heme binding and folding

We studied the effect of deleted and circularly permuted mutations in sperm whale myoglobin and present here results on three classes of mutants: (i) a deletion mutant, Mb(1)(-)(99), in which the C-terminal helices, G and H, were removed; (ii) two circular permutations, Mb-B_GHA, in which helix B is N-terminal and helix A is C-terminal, and Mb-C_GHAB, in which helix C is N-terminal and helices A and B are C-terminal; and (iii) a deleted circular permutation, Mb-HAB_F, in which helix H is N-terminal, helix F is C-terminal, and helix G is deleted. The conformational characteristics of the apo and holo forms of these mutants were determined at neutral pH, by spectroscopic and hydrodynamic methods. The apo form of the deleted and permuted mutants exhibited a stronger tendency to aggregate and had lower ellipticity than the wild type. The mutants retained the ability to bind heme, but only the circularly permuted holoproteins had native-like heme binding and folding. These results agree with the theory that myoglobin has a central core that is able to bind heme, but also indicate that the presence of N- and C-terminal helices is necessary for native-like heme pocket formation. Because the holopermuteins were less stable than the wild-type protein and aggregated, we propose that the native position of the N-terminus is important for the precise structural architecture of myoglobin.