Histidine Linkage Research Articles

Facile Heme Vinyl Posttranslational Modification in a Hemoglobin

Iron-protoporphyrin IX, or b heme, is utilized as such by a large number of proteins and enzymes. In some cases, notably the c-type cytochromes, this group undergoes a posttranslational covalent attachment to the polypeptide chain, which adjusts the physicochemical properties of the holoprotein. The hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803 (GlbN), contrary to the archetypical hemoglobin, modifies its b heme covalently. The posttranslational modification links His117, a residue that does not coordinate the iron, to the porphyrin 2-vinyl substituent and forms a hybrid b/c heme. The reaction is an electrophilic addition that occurs spontaneously in the ferrous state of the protein. This apparently facile type of heme modification has been observed in only two cyanobacterial GlbNs. To explore the determinants of the reaction, we examined the behavior of Synechocystis GlbN variants containing a histidine at position 79, which is buried against the porphyrin 4-vinyl substituent. We found that L79H/H117A GlbN bound the heme weakly but nevertheless formed a cross-link between His79 Nε2 and the heme 4-Cα. In addition to this linkage, the single variant L79H GlbN also formed the native His117-2-Cα bond yielding an unprecedented bis-alkylated protein adduct. The ability to engineer the doubly modified protein indicates that the histidine-heme modification in GlbN is robust and could be engineered in different local environments. The rarity of the histidine linkage in natural proteins, despite the ease of reaction, is proposed to stem from multiple sources of negative selection.

Resonance Raman spectroscopic studies of hydroperoxo derivatives of cobalt-substituted myoglobin

Recent progress in generating and stabilizing reactive heme protein enzymatic intermediates by cryoradiolytic reduction has prompted application of a range of spectroscopic approaches to effectively interrogate these species. The impressive potential of resonance Raman spectroscopy for characterizing such samples has been recently demonstrated in a number of studies of peroxo- and hydroperoxo-intermediates. While it is anticipated that this approach can be productively applied to the wide range of heme proteins whose reaction cycles naturally involve these peroxo- and hydroperoxo-intermediates, one limitation that sometimes arises is the lack of enhancement of the key intraligand ν(O–O) stretching mode in the native systems. The present work was undertaken to explore the utility of cobalt substitution to enhance both the ν(Co–O) and ν(O–O) modes of the CoOOH fragments of hydroperoxo forms of heme proteins bearing a trans–axial histidine linkage. Thus, having recently completed RR studies of hydroperoxo myoglobin, attention is now turned to its cobalt-substituted analogue. Spectra are acquired for samples prepared with 16O 2 and 18O 2 to reveal the ν(M–O) and ν(O–O) modes, the latter indeed being observed only for the cobalt-substituted proteins. In addition, spectra of samples prepared in deuterated solvents were also acquired, providing definitive evidence for the presence of the hydroperoxo-species.

Bidentate ligation of heme analogues; novel biomimetics of peroxidase active site.

The multifunctional nature of proteins that have iron-heme cofactors with noncovalent histidine linkage to the protein is controlled by the heme environment. Previous studies of these active-site structures show that the primary difference is the length of the iron-proximal histidine bond, which can be controlled by the degree of H-bonding to this histidine. Great efforts to mimic these functions with synthetic analogues have been made for more than two decades. The peroxidase models resulted in several catalytic systems capable of a large range of oxidative transformations. Most of these model systems modified the porphyrin ring covalently by directly binding auxiliary elements that control and facilitate reactivity; for example, electron-donating or -withdrawing substituents. A biomimetic approach to enzyme mimicking would have taken a different route, by attempting to keep the porphyrin ring system unaltered, as close as possible to its native form, and introducing all modifications at or close to the axial coordination sites. Such a model system would be less demanding synthetically, would make it easy to study the effect of a single structural modification, and might even provide a way to probe effects resulting from porphyrin exchange. We introduce here an alternative model system based on these principles. It consists of a two component system: a bis-imidazolyl ligand and an iron-porphyrin (readily substituted by a hemin). All modifications were introduced only to the ligand that engulfs the porphyrin and binds to the iron's fifth and sixth coordination sites. We describe the design, synthesis, and characterization of nine different model compounds with increased complexity. The primary tool for characterizing the environment of each complex Fe(III) center was the Extended X-ray Absorption Fine Structure (EXAFS) measurements, supported by UV/Vis, IR, and NMR spectroscopy and by molecular modeling. Introduction of asymmetry, by attaching different imidazoles as head groups, led to the formation of two axial bonds of different length. Addition of H-bonds to one of the imidazoles in an advanced model increased this differentiation and expanded the porphyrin ring. These complexes were found to be almost identical in structure to peroxidase active sites. Similarly to the peroxidases and other synthetic models, these compounds stabilize the green, compound I-like intermediate, and catalyze the oxidation of organic substrates.

Insights into the Functional Role of the Tyrosine−Histidine Linkage in Cytochrome c Oxidase

Insights into the Functional Role of the Tyrosine−Histidine Linkage in Cytochrome <i>c</i> Oxidase

Evidence for Damped Hemoglobin Dynamics in a Room Temperature Trehalose Glass

Upon photodissociation of its ligand, COHbA exhibits a wide range of nonequilibrium relaxation phenomena that start within a fraction of a picosecond and extend out to tens of microseconds. In addition, equilibrium fluctuations of the protein result in conformational averaging. All of these dynamics can have an impact on ligand rebinding. In an effort to better understand the relationship between conformational dynamics and ligand-binding reactivity, COHbA was embedded in a room temperature trehalose sugar glass (Hagen et al. Science 1995, 269, 959) in order to uncouple solvent motions from protein dynamics as well as reduce the amplitude of large-scale protein conformational fluctuations. Time-resolved resonance Raman spectroscopy and ligand-rebinding kinetics show that the trehalose glass does not impede the initial fast relaxation of the iron−histidine linkage, but does dramatically impede conformational averaging and completely eliminates ligand escape at all temperatures from 140 K to room temperature. Fluorescence measurements indicate that in the trehalose glass the picosecond tryptophan lifetimes are nearly unchanged, but there is a complete absence of the nanosecond fluorescence decay (observed in aqueous solutions), which is replaced by a decay of ∼700 ps. This change in the fluorescence decay is ascribed to a significant decrease in the structural dynamics that normally allow transient opening of the distal heme pocket.

Resonance Raman Study of Cyanide-Ligated Horseradish Peroxidase.Detection of Two Binding Geometries and Direct Evidence for the "Push-Pull" Effect

Resonance Raman spectroscopy has been employed to investigate the structure of cyanide adducts of the basic isoenzymes of horseradish peroxidase (HRP) in the pH range 5.5-12.5. Evidence for the binding of cyanide in two forms, characterized by the reversal of ordering of the Fe-CN stretching and Fe-C-N bending vibrations, is observed. Moreover, it is shown that both conformers exhibit an acid-alkaline transition in the pH range employed. In the first conformer, the Fe-C-N linkage is essentially linear, exhibiting axial Fe-CN stretching and Fe-C-N bending frequencies at 453 and 405 cm-1, respectively (at pH 5.5) (Lopez-Garriga et al., 1990). In the second conformer, the Fe-C-N fragment is bent, and the axial stretching and bending modes have been identified at 360 and 422 cm-1 at pH 5.5. At pH 12.5, the v[Fe-CN] stretching mode of the linear conformer shifts down by 9 cm-1 to 444 cm-1 while the bending frequency remains unchanged. For the bent conformer at this pH, the stretching mode shifts to 355 cm-1 (-5 cm-1), and the bending vibration shifts slightly to lower frequency by 2 cm-1 to 420 cm-1. The observed pH-dependent shift of the v[Fe-CN] stretching mode of the linear conformer is attributed to the direct effect of deprotonation of a distal-side amino acid residue while the shift of v[Fe-CN] of the bent conformer is most reasonably ascribable to indirect alteration of the iron-proximal histidine linkage induced by the distal-side deprotonation, a spectral response which reflects a protein-coupled "push-pull" mechanism for heterolytic O-O bond cleavage.

A possible new control mechanism suggested by resonance Raman spectra from a deep ocean fish hemoglobin

The rattail fish, Coryphaenoides armatus, lives at ocean depths of 3000 m. As an adaptation for pumping oxygen into the swim bladder against the extreme pressures at the ocean bottom, the hemoglobin from this fish at low pH exhibits an extraordinarily low affinity for ligands. In this study, continuous wave and time-resolved Raman techniques are used to probe the binding site in this hemoglobin. The findings show an association between the low-affinity material and a highly strained heme-proximal histidine linkage. The transient Raman studies reveal differences in the protein structural dynamics at pH 6 and 8. The emerging picture derived from both this and earlier studies is that in vertebrate hemoglobins the heme-proximal histidine linkage represents a key channel through which species- and solution-dependent variations in the globin are communicated both statically and dynamically to the heme to produce an extensive range of ligand binding properties. Also presented is a new model that relates both intensity and frequency of the resonance Raman band involving the iron-proximal histidine stretching mode to specific protein controlled structural degrees of freedom. There emerges from this model a mechanism whereby modifications in the proximal heme pocket can further reduce the affinity of an already highly strained T state structure of hemoglobin.

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins.

A key feature of the Perutz stereochemical model for cooperativity in hemoglobin is a strong coupling between quaternary structure and the spin state of the heme iron [Perutz, M. F. (1979) Annu. Rev. Biochem. 48, 327-386]. While this coupling appears to be present for carp azide methemoglobin, it should also be present for all liganded forms of human methemoglobin that exhibit a thermal high-spin in equilibrium low-spin equilibrium. To test this hypothesis, we have measured the changes in spin equilibria upon conversion of six mixed-spin forms of human methemoglobin from the R (high-affinity) to the T (low-affinity) quaternary structure by addition of inositol hexaphosphate. These experiments were done with a sensitive superconducting magnetic susceptibility instrument on solutions at 20 degrees C in 20 mM maleate buffer, pH 6. The data show zero or small increases in high-spin content upon switching from R to T, changes that are equivalent to a relative stabilization of the high-spin form by only 0-300 cal mol-1 heme-1. These changes in energy are far less than the 1200 cal mol-1 heme-1 predicted from the Perutz stereochemical model [Cho, K. C., & Hopfield, J. J. (1979) Biochemistry 18, 5826-5833]. That is, these data do not support a view that the low affinity of the T state is due to restraints acting through the iron-proximal histidine linkage. The mechanistic implications of these results and the differences between species and ferric ligands are discussed.

The iron-proximal histidine linkage and protein control of oxygen binding in hemoglobin. A transient Raman study.

Time-resolved Raman studies have shown that communication between the heme oxygen binding sites and the surrounding globin occurs through the iron-proximal histidine linkage. By comparing the frequency of the Fe-His stretching mode in equilibrium deoxy- and photoinduced transient deoxyhemoglobins, we have found that ligand binding induces protein structural changes that strengthen the Fe-His linkage. The extent of this increase is observed to depend upon the quaternary state. This dependence is reflected in the restricted set of values observed in transient and equilibrium studies for the frequency of the Fe-His stretching mode in both R and T state deoxyhemoglobins. For T state hemoglobins the frequency increases from 215 cm-1 to approximately 222 cm-1 in going from the stable to the nanosecond transient deoxy species. The corresponding change for R state human hemoglobins is from approximately 222 cm-1 to approximately 230 cm-1. Studies on transients derived from Fe-Co hybrid hemoglobins reveal no subunit heterogeneity associated with the R state transient (230 cm-1) at high pH. We also observe that perturbations of the protein known to destabilize the structure of ligand-bound R state hemoglobins are associated with a decrease in the frequency of the iron-proximal histidine stretching mode in the corresponding transient species. Such effects can originate from changes in either quaternary state or solution conditions as well as species-specific variations in protein structure. These results indicate that modulation of the Fe-His linkage could be a general mechanism for regulating ligand binding properties in hemoglobin. A direct connection between ligand binding and the Fe-His bond is suggested from our finding that the structural parameter(s) regulating the barrier height for germinate recombination is likely to be the same as the one(s) modulating the frequency of the Fe-His stretching mode in the transient deoxy species. Based on the recent conclusion that the variation in the tilt of histidine with respect to the heme plane is primarily responsible for the spectrum of frequencies observed for the Fe-His stretching mode we have examined a model in which protein regulation of binding occurs via a protein-induced change in the histidine tilt.

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.

Gene-enzyme relationships in histidine biosynthesis in Bacillus subtilis.

Mutants for 9 of the 10 steps in histidine biosynthesis have been isolated and identified by enzyme assay. Each locus has been mapped in relation to the aro cluster and to other histidine loci by deoxyribonucleic acid-mediated transformation. The genes which code for enzymes 3, 6, and 8 of the pathway are linked to the aro cluster. A major histidine linkage group is composed of the genes which specify enzymes 1, 2, 5, 7, and 10. The locus which codes for step 9 of the pathway is unlinked to any other identified his loci. The major histidine cluster is loosely linked to cysB and is unlinked to any of the loci concerned with aromatic amino acid biosynthesis.

Genetic analysis, by means of transformation, of histidine linkage groups in Bacillus subtilis.

Genetic analysis, by means of transformation, of histidine linkage groups in Bacillus subtilis.