Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4.

The nitrophorins are a family of proteins that use ferric heme to transport nitric oxide (NO) from the salivary glands of blood-sucking insects to their victims, resulting in vasodilation and reduced blood coagulation. We have refined atomic resolution structures of nitrophorin 4 (NP4) from Rhodnius prolixus complexed with NO (1.08 A) and NH(3) (1.15 A), yielding a highly detailed picture of the iron coordination sphere. In NP4-NO, the NO nitrogen is coordinated to iron (Fe-N distance = 1.66 A) and is somewhat bent (Fe-N-O angle = 156 degrees ), with bending occurring in the same plane as the proximal histidine ring. The Fe(NO)(heme)(His) coordination geometry is unusual but consistent with an Fe(III) oxidation state that is stabilized by a highly ruffled heme. Heme ruffling occurs in both structures, apparently due to close contacts between the heme and leucines 123 and 133, but increases on binding NO even though the steric contacts have not changed. We also report the structure of NP4 in complexes with histamine (1.50 A) and imidazole (1.27 A). Unexpectedly, two mobile loops that rearrange to pack against the bound NO in NP4-NO, also rearrange in the NP4-imidazole complex. This conformational change is apparently driven by the nonpolar nature of the NO and imidazole (as bound) ligands. Taken together, the desolvation of the NO binding pocket through a change in protein conformation, and the bending of the NO moiety, possibly through protein-assisted heme ruffling, may lead to a nitrosyl-heme complex that is unusually resistant to autoreduction.

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus.

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

Self-assembly of mesoscopically ordered chromatic polydiacetylene/silica nanocomposites.

Nature abounds with intricate composite architectures composed of hard and soft materials synergistically intertwined to provide both useful functionality and mechanical integrity. Recent synthetic efforts to mimic such natural designs have focused on nanocomposites, prepared mainly by slow procedures like monomer or polymer infiltration of inorganic nanostructures or sequential deposition. Here we report the self-assembly of conjugated polymer/silica nanocomposite films with hexagonal, cubic or lamellar mesoscopic order using polymerizable amphiphilic diacetylene molecules as both structure-directing agents and monomers. The self-assembly procedure is rapid and incorporates the organic monomers uniformly within a highly ordered, inorganic environment. Polymerization results in polydiacetylene/silica nanocomposites that are optically transparent and mechanically robust. Compared to ordered diacetylene-containing films prepared as Langmuir monolayers or by Langmuir-Blodgett deposition, the nanostructured inorganic host alters the diacetylene polymerization behaviour, and the resulting nanocomposite exhibits unusual chromatic changes in response to thermal, mechanical and chemical stimuli. The inorganic framework serves to protect, stabilize, and orient the polymer, and to mediate its function. The nanocomposite architecture also provides sufficient mechanical integrity to enable integration into devices and microsystems.

Porphyrin interactions with wild-type and mutant mouse ferrochelatase.

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes Fe(2+) chelation into protoporphyrin IX. Resonance Raman and UV-vis absorption spectroscopies of wild-type and engineered variants of murine ferrochelatase were used to examine the proposed structural mechanism for iron insertion into porphyrin. The recombinant variants (i.e., H207N and E287Q) are enzymes in which the conserved amino acids histidine-207 and glutamate-287 of murine ferrochelatase were substituted with asparagine and glutamine, respectively. Both of these residues are at the active site of the enzyme as deduced from the Bacillus subtilis ferrochelatase three-dimensional structure. On the basis of changes in the UV-vis absorption spectrum, addition of free-base or metalated porphyrins to wild-type ferrochelatase and H207N variant yields a 1:1 complex, most likely a monomeric protein-bound species at the active site. In contrast, the addition of porphyrin (either free base or metalated) to E287Q is substoichiometric, as this variant retains bound porphyrin in the active site during isolation and purification. The specificity of porphyrin binding is confirmed by the narrowing of the structure-sensitive lines and the vinyl vibrational mode in the resonance Raman spectra. Shifts in the resonance Raman lines of free-base and metalated porphyrins bound to the wild-type ferrochelatase indicate a nonplanar distortion of the porphyrin macrocycle. However, the magnitude of the distortion cannot be determined without first defining the specific type of deformation. Significantly, the extent of the nonplanar distortion varies in the case of H207N- and E287Q-bound porphyrins. In fact, resonance Raman spectral decompositions indicate a homogeneous ruffled deformation for the nickel protoporphyrin bound to the wild-type ferrochelatase, whereas both planar and ruffled conformations are present for the H207N-bound porphyrin. Perhaps more revealing is the unusual resonance Raman spectrum of the endogenous E287Q-bound porphyrin, which has the structure-sensitive lines greatly upshifted relative to those of the free-base protoporphyrin in solution. This could be interpreted as an equilibrium between protein conformers, one of which favors a highly distorted porphyrin macrocycle. Taken together, these findings suggest that distortion occurs in murine ferrochelatase for some porphyrins, even without metal binding, which is apparently required for the yeast ferrochelatase.

The quantum mixed-spin heme state of barley peroxidase: A paradigm for class III peroxidases.

Electronic absorption and resonance Raman (RR) spectra of the ferric form of barley grain peroxidase (BP 1) at various pH values, at both room temperature and 20 K, are reported, together with electron paramagnetic resonance spectra at 10 K. The ferrous forms and the ferric complex with fluoride have also been studied. A quantum mechanically mixed-spin (QS) state has been identified. The QS heme species coexists with 6- and 5-cHS hemes; the relative populations of these three spin states are found to be dependent on pH and temperature. However, the QS species remains in all cases the dominant heme spin species. Barley peroxidase appears to be further characterized by a splitting of the two vinyl stretching modes, indicating that the vinyl groups are differently conjugated with the porphyrin. An analysis of the currently available spectroscopic data for proteins from all three peroxidase classes suggests that the simultaneous occurrence of the QS heme state as well as the splitting of the two vinyl stretching modes is confined to class III enzymes. The former point is discussed in terms of the possible influences of heme deformations on heme spin state. It is found that moderate saddling alone is probably not enough to cause the QS state, although some saddling may be necessary for the QS state.

Resonance Raman investigation of nickel microperoxidase-11.

Resonance Raman and UV-visible absorption spectra show that nickel(II) microperoxidase-11 (NiMP-11) is four-coordinate in aqueous solution in the pH range from 1.0 to 13.0. In aqueous solutions of NiMP-11 in the absence of cetyltrimethylammonium bromide (CTAB), NiMP-11 is aggregated. In CTAB micellar solutions, where aggregation of NiMP-11 does not occur, the Raman spectra of NiMP-11 are similar to that of nickel(II) cytochrome c (NiCyt-c). The presence of the peptide segment shifts the equilibrium heavily in favor of the nonplanar form, just as does the entire protein component in the case of NiCyt-c. This further elucidates the structural mechanism by which the protein segment ruffles the heme, most likely modulating the redox potential as indicated for the cytochromes c3 [Ma, J.-G., et al. (1998) Biochemistry 37, 12431-12442]. Furthermore, the hydrophobic environment that is provided by the CTAB micelle is found to be crucial to the native folding of the pentapeptide and formation of two hydrogen bonds in the peptide backbone. These two H-bonds act to contract the peptide segment exerting the force on the macrocycle that causes the ruffling and makes the redox potential more negative than if the heme were to remain planar. The structure of the heme and pentapeptide may also be associated with redox-linked triggering of the formation and release of cytochrome-protein complexes.

The structural origin of nonplanar heme distortions in tetraheme ferricytochromes c3.

Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal-coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c3. NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (nu4, nu3, and nu2) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c3 (Norway 4 and 9974) than for the Desulfovibrio (D.) gigas, D. vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the nu3 and nu10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins.

Protein-induced changes in nonplanarity of the porphyrin in nickel cytochrome c probed by resonance Raman spectroscopy.

The influence of the protein on the nonplanarity of the macrocycle for nickel(II)-reconstituted cytochrome c (NiCyt-c) has been investigated with pH-dependent resonance Raman and UV-visible absorption spectroscopy and molecular mechanics calculations. The spectra reveal that NiCyt-c near neutral pH has axially coordinated Ni, but below pH 3 and above pH 12, four-coordinate species predominate. The shape of the structure-sensitive Raman line nu10 of NiCyt-c is asymmetric and broad and it changes with pH. This broad line can be decomposed well into at least two sublines, a low-frequency line that results from a nonplanar conformer and a high-frequency line that arises from a nearly planar conformer. Upon lowering the pH from 3.0 to 1.0, the amount of the nonplanar conformer decreases relative to that of the planar conformer. The decreased nonplanarity can be accounted for in terms of the disruption of a hydrogen-bonding network in the peptide backbone upon lowering the pH. Molecular mechanics (MM) calculations on iron(III) and nickel(II) microperoxidase 5 (MP-5) as well as some model heme derivatives have been carried out in order to locate the part of the protein that causes the heme distortion observed in the X-ray crystal structures of cytochromes c. The energy-optimized structures of MP-5 and the model compounds were analyzed using the normal-coordinate structural decomposition method to specify and quantify the out-of-plane macrocyclic distortions. MM calculations for MP-5 show that two hydrogen bonds formed between the amide groups in the peptide backbone are important in maintaining the ruffled deformation of the macrocycle. All evidence presented supports the hypothesis that the nonplanar distortion of the porphyrin of cytochromes c is largely maintained by a relatively small protein segment including the cysteines, the amino acids between the cysteines, and the adjacent histidine ligand. Hydrogen bonding within the backbone of this segment is important in maintaining the conformation of the peptide that induces the porphyrin distortion.

Conservation of the conformation of the porphyrin macrocycle in hemoproteins.

The out-of-plane distortions of porphyrins in hemoproteins are characterized by displacements along the lowest-frequency out-of-plane normal coordinates of the D4h-symmetric macrocycle. X-ray crystal structures are analyzed using a computational procedure developed for determining these orthogonal displacements. The x-ray crystal structures of the heme groups are described within experimental error, using the set composed of only the lowest frequency normal coordinate of each out-of-plane symmetry type. That is, the distortion is accurately simulated by a linear combination of these orthonormal deformations, which include saddling (B2u), ruffling (B1u), doming (A2u), waving (Eg), and propellering (A1u). For example, orthonormal structural decomposition of the hemes in deoxymyoglobins reveals a predominantly dom heme deformation combined with a smaller wav(y) deformation. Generally, the heme conformation is remarkably similar for proteins from different species. For cytochromes c, the conformation is conserved as long as the amino acids between the cysteine linkages to the heme are homologous. Differences occur if this short segment varies in the number or type of residues, suggesting that this small segment causes the nonplanar distortion. Some noncovalently linked hemes like those in the peroxidases also have highly conserved characteristic distortions. Conservation occurs even for some proteins with a large natural variation in the amino acid sequence.

Isolation and characterization of vibrational spectra of individual heme active sites in cytochrome bc1 complexes from Rhodobacter capsulatus.

Resonance Raman spectra of bc1 complexes and isolated c1 subunit from Rhodobacter capsulatus have been obtained using a variety of excitation wavelengths. Spectra obtained via Q-band excitation of bc1 complexes in different redox states were separated to yield the individual vibrational spectra of each of the three heme active sites. Hemes bH and c1 exhibit vibrational spectra typical of b- and c-type hemes, respectively. In contrast, the spectrum of heme bL is anomalous with respect to those of other hemes b. The isolated spectra were also used to assess the effects of inhibitor binding on the local structural environments of the hemes. Neither antimycin nor myxothiazol binding produces dramatic structural perturbations at the hemes. Heme c1 is completely unaffected by the presence of either inhibitor. The vibrational spectra of hemes bH and bL are slightly altered by antimycin and myxothiazol binding, respectively.

Conserved nonplanar heme distortions in cytochromes c.

A nonplanar distortion of the heme of c-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available high-resolution X-ray crystal structures. This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion. This asymmetry in the heme distortion is also conserved. For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed. Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (> or = 2.00 A) is energetically unfavorable and can occur only in the presence of significant environmental perturbations. Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochrome c, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure. It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties of c-type cytochromes.

Coordination chemistry of F430. Axial ligation equilibrium between square-planar and bis-aquo species in aqueous solution.

X-ray absorption spectroscopic characterization of axial ligand coordination to factor F430, the nickel-tetrapyrrole cofactor of the S-methyl-coenzyme M (CH3SCoM) methyl reductase enzyme from methanogenic bacteria, is presented. The nickel of isolated F430 is hexacoordinate at 10 K in aqueous solution (as is the enzyme-bound cofactor), whereas the epimerized and ring-oxidized derivatives of F430 have four-coordinate nickel. Reduction of the ring-oxidized derivative, F560, with dithionite yields F430 in its native configuration, with axial ligands indistinguishable from those present when the cofactor is obtained directly from the holoenzyme. Thus, we conclude that the axial ligands to F430 in aqueous solution are water molecules. Analysis of the nickel extended x-ray absorption fine structure is consistent with this conclusion. Resonance Raman spectra obtained at room temperature contain features characteristic of both 4- and 6-coordinate forms of the cofactor. We have found that the resonance Raman, optical, and x-ray absorption spectra of aqueous solutions of F430 are temperature-dependent due to a ligand-binding equilibrium involving the square-planar and 6-coordinate bis-aquo forms of the cofactor. At low temperatures (less than 250 K) the 6-coordinate form predominates, whereas higher temperature solutions contain both 4- and 6-coordinate species in a dynamic equilibrium. Similar behavior is observed in other weakly coordinating solvents such as methanol and ethanol. The 4-coordinate form is predominant in solvents with strong electron-withdrawing substituents such as 2,2,2-trifluoroethanol and 2-mercaptoethanol. The relevance of this facile ligand exchange to the active site structure and enzymatic mechanism of the parent enzyme is discussed.

Structural and spectroscopic characterization of exogenous ligand binding to isolated factor F430 and its configurational isomers.

Binding of axial ligands to the nickel(II) of isolated factor F430 from the methyl reductase enzyme of Methanobacterium thermoautotrophicum is demonstrated. Evidence of bis-ligand coordination is obtained from the x-ray absorption, optical, and resonance Raman spectral characterization of F430 and its 12,13-diepimeric isomer in the presence of a large excess of cyanide, pyridine, or 1-methylimidazole. Significant broadening and 5-10-nm red shifts of the main 430-nm optical absorption band and shifts of up to 30 cm-1 for the high-frequency Raman lines are observed upon coordination of these axial ligands. The Raman spectra of native F430 and the diepimer with a particular axial ligand are nearly identical. Nickel x-ray absorption edge spectra of the diepimer in the absence and presence of these exogenous ligands are indicative of conversion from a square-planar to a tetragonally distorted octahedral geometry. Analyses of the nickel extended x-ray absorption fine structure data for the ligated diepimer complexes yield detailed structural information for these complexes. Implications of these data with respect to the enzymatic mechanism and the structure of the enzyme-bound factor are discussed.

A resonance Raman study of the binding of ethanol and methanol to ferrihemoglobin.

The interactions of ethanol and methanol with ferrihemoglobin were examined using resonance Raman spectroscopy. After binding either alcohol, the low-frequency resonance Raman spectra of human ferrihemoglobin are almost identical to the unperturbed spectrum except for shifts in the 309 cm-1 band to higher frequency by as much as 8 cm-1. The ethanol-induced shift is greater than that with methanol even though complex formation was less for ethanol than methanol. The spectral changes imply a site-specific, similar binding of these alcohols to ferrihemoglobin which may involve steric interactions. Possible assignments of the 309 cm-1 band to structural features as well as potential mechanisms of the alcohol-induced spectral changes are discussed.

Heme-linked ionizations of myeloperoxidase detected by Raman difference spectroscopy. A comparison with plant and yeast peroxidases.

The pH-dependence of the oxidation state marker line v4 of human leucocyte myeloperoxidase is determined in the absence of chloride using Raman difference spectroscopy (RDS). A transition in the frequency of v4 with pK of 4.2 +/- 0.3 is found. The pK compares favorably with that previously determined by spectrophotometric titration and kinetic studies. The shift in v4 across the transition is -1.3 cm-1. The shift in v4 and other Raman marker lines indicates enhanced pi charge in the chlorin ring below the transition. The low frequencies of the oxidation state marker lines indicate that a structural change occurs near the chromophore, which results in the formation of a more pi-charge donating protein environment for the chlorin ring at low pH. The Raman results are discussed in terms of a proposed catalytic control mechanism based on charge stabilization of the energy of ring charge-depleted ferryl intermediates of the reaction with peroxide. The myeloperoxidase findings are compared with similar RDS results for ferrous horseradish peroxidase and ferric cytochrome c peroxidase.

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode.

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)

Heme-linked ionizations in horseradish peroxidase detected by Raman difference spectroscopy.

Heme-linked ionizations of the acidic and basic isoenzymes of ferrous horseradish peroxidase influence both the Fe-histidine stretching mode and the oxidation-state marker line. First, Raman difference spectroscopy of horseradish peroxidase confirms earlier work showing that v(Fe-His) undergoes a transition in frequency with a pK that is characteristic of the enzyme's functional properties. The Fe-histidine mode shifts by about 2.5-3.0 cm-1 for horseradish peroxidase C and by about 6 cm-1 for the acidic isoenzyme. Further, we find that the oxidation-state marker line v4 also exhibits a transition with the same pK. For horseradish peroxidase C the shift in v4 is 0.4 cm-1 and the pK is 7.1 +/- .5, in good agreement with the pK found by other techniques. Shifts in these two Raman lines are correlated for the pK 7.1 transition and attain their highest frequency at low pH. The correlation is in marked contrast with R/T shifts in hemoglobins for which delta v(Fe-His) and delta v4 are also linearly related but shift in opposite directions. The shift in v4 suggests a mechanism for pH control of catalytic function based on ring pi-charge density effects on the energy of charge-depleted high oxidation-state intermediates. A second transition in v4 (delta v4 = 2.6 cm-1) with a pK of 10.0 is interpreted in terms of a change in ligation and spin state.

A resonance Raman study of Ambystoma tigrinum hemoglobins: evidence for intraspecies hemepocket variations.

Using resonance Raman difference spectroscopy, the Raman-active vibrational modes of hemoglobins from adult, neotenic, and larval forms of the salamander, Ambystoma tigrinum have been compared to each other and to human hemoglobin. The local heme environment of the adult and neotenic proteins were identical and differed from that of the larval protein. Differences were observed in modes sensitive to porphyrin pi electron density and axial ligation. Systematic differences were also observed between human and adult salamander hemoglobins particularly in modes sensitive to the heme vinyl environment. The relationship between these environmental differences, oxygen binding affinity, and the effects of allosteric modulators are discussed.