pH-dependent changes in the tryptophan and porphyrin fluorescence of the apomyoglobin complex with protoporphyrin IX and methemoglobin

The fluorescence of protoporphyrin IX (PPIX) complexed with sperm whale apomyoglobin as well as the tryptophan fluorescence of this complex and of metmyoglobin within the pH range of 3.5-13 was studied. It was shown that an increase in pH from 5.3 to 10.8 does not influence the fluorescence of PPIX in the complex and causes no essential changes in the fluorescence of Trp residues, which occur at more acidic and, correspondingly, alkaline pH values simultaneously with the protein denaturation. This is accompanied by a sharp increase in the quantum yield of tryptophan fluorescence due to dissociation of PPIX from the complex. Similar changes are observed in metMb at pH less than 4.3 and greater than 12 which is concomitant with absorption changes in the Soret band, thus indicating a higher stability of metMb towards the acid and alkaline denaturation as compared to the complex. In both cases, a slight alteration in the shape of the tryptophan fluorescence spectrum is observed, which precedes alkaline denaturation of the Mb molecule and is probably due to changes in the conformation of the N-terminal region caused by the break of the salt bridges stabilizing the native structure of the protein.     

Structural features of the protoporphyrin-apomyoglobin complex: a proton NMR spectroscopy study

The structural properties of the complex formed by apomyoglobin and protoporphyrin IX (des-iron myoglobin) were studied to probe the influence of iron-to-histidine coordination on the native myoglobin fold and the heme binding site geometry. Standard two-dimensional proton nuclear magnetic resonance spectroscopy methods were applied to identify porphyrin and protein signals. A pronounced spectral resemblance between carbonmonoxymyoglobin and des-iron myoglobin was noticed that could be exploited to assign a number of resonances by nuclear Overhauser spectroscopy. Protoporphyrin IX was determined to bind in the same orientation as the heme. Most residues in contact with the prosthetic group were found in the holomyoglobin conformation. Several tertiary structure features were also characterized near the protein termini. It was concluded that the protoporphyrin-apomyoglobin interactions are capable of organizing the binding site and the unfolded region of the apoprotein into the native holoprotein structure.     

Fluorescence study of conformational transitions in the structure of myoglobin

Fluorescence studies of myoglobin and Mb-like structures, apomyoglobin and the complex of apo-Mb with protoporphyrin IX, reveal both the similarity between them, which is due to a common type of polypeptide chain folding, and the distinctions imposed by the influence of the prosthetic group. Close resemblance of structures of holomyoglobin and its metal-free analog, PPIX--apo-Mb, points to a key role of specific interactions between the protein and the protoporphyrin macrocycle rather than the Fe-protein bond in the formation of Mb-like structures. In PPIX--apo-Mb, both the hydrophobic core and the important ionic bonds between different structural elements () stabilizing the Mb structure are almost completely retained. The bond between Fe and proximal His-F8 allows additional integration of the structures of the heme cavity and the myoglobin molecule as a whole, providing its functional activity and highly cooperative conformational transitions. In all the myoglobin-like structures studied, a certain relationship is found between conformational states of the , the heme cavity, and the N-terminal part of the molecule. This is probably due to variations in the mutual orientation of the ABCDE and FGH helical domains, depending on the interactions between the protein, the prosthetic group, and the ligand in the heme crevice. The correlation between conformations of the N-terminal and heme regions found at a level of the globin tertiary structure is very important for understanding the mechanisms of homo- and heterotropic regulation in tetrameric hemoglobins.     

Copper(II) protoporphyrin IX as a reporter group for the heme environment in myoglobin.

Copper(II) protoporphyrin IX has been introduced into apomyoglobin, and its utility as a reporter group of the heme environment has been examined. The Soret and visible absorption bands and electron spin resonance spectrum show that the Cu(II) is five coordinate, probably through coordination to the F-8 proximal histidine. The resonance Raman spectrum does not indicate any appreciable distortion from the solution conformation of copper(II) protoporphyrin IX dimethyl ester in CS2. The ultraviolet circular dichroism shows no alteration of the helical content of the globin from that of metmyoglobin. The circular dichroism of the porphyrin transitions suggests that the packing of the amino acid side chains around the porphyrin is different than that in the native metmyoglobin.     

Spectral studies of horse heart porphyrin cytochrome c

Removal of the heme iron from cytochrome c to generate porphyrin cytochrome c relieves the quenching of porphyrin fluorescence and enhances the fluorescence of the single tryptophan residue and the 4 tyrosine residues. The intensity of the porphyrin fluorescence is not perturbed by denaturation of the protein at neutral pH using either urea or guanidine hydrochloride. However, the amplitude of tryptophan fluorescence is increased by these denaturants from 5 to about 85% of a model tryptophan residue using solutions of 2 microM tryptophan. In contrast to cytochrome c, the tryptophan fluorescence amplitude of denatured porphyrin cytochrome c is independent of pH over the range pH 3.0 to 7.4. Acidification of solutions of either native or denatured porphyrin cytochrome c markedly alters both the visible absorbance and fluorescence of the protein consistent with protonation of two pyrrole nitrogens on the porphyrin. Preliminary analysis of the spectral changes occurring in the acid transition suggests the presence of an intermediate form having only one of these two pyrrole nitrogens protonated.     

Fluctuation domains in myoglobin. Fluorescence quenching studies

The dynamics of two domains in the myoglobin molecule, close to the heme and inside the protein medium including the surface, are investigated through the study of the fluorescence oxygen quenching of two probes imbedded in the heme pocket: zinc protoporphyrin IX (with a fluorescence lifetime of 2.1 ns) and metal-free protoporphyrin IX (with a fluorescence lifetime of 17.8 ns).     

Structural characterization of non-native states of sperm whale myoglobin in aqueous ethanol or 2,2,2-trifluoroethanol media

The effects of aqueous ethanol or 2,2,2-trifluoroethanol media on the structure of sperm whale myoglobin have been investigated by absorption, CD, and NMR spectra. The structural properties of myoglobin such as heme environments, helix contents, protein folding, and interactions between heme and the protein moiety have been sharply manifested in these spectra. The characterization demonstrated that alcohol-induced conformational change of myoglobin depends on the nature of alcohol and its concentration. It was shown for the first time that, upon the alcohol-induced denaturation of myoglobin, heme is released from partially denatured protein of which helix contents is altered by only about 20% relative to that of native state. Myoglobin has shown to unfold and refold reversibly by controlling the alcohol concentration. Novel methods for the preparation of apomyoglobin and in situ reconstitution of apomyoglobin with heme, based on the alcohol-induced denaturation of the protein, were presented.     

Structure and function of the myoglobin containing octaethylhemin as a prosthetic group

Spectrophotometric titration of ferric octaethylporphyrin (OEP) with apomyoglobin revealed their 1:1 complex formation. Proton NMR spectrum of the OEP-reconstituted deoxymyoglobin exhibits an exchangeable peak from the proximal F8 histidine at 78.5 ppm, indicating the incorporation of iron OEP into the heme cavity to form the Fe-N(His-F8) bond. OEP metmyoglobin without external ligand has an iron-bound water that deprotonates above pH 7.8. Affinities of the aquometmyoglobin for several ionic ligands were comparable with those of native metmyoglobin. Deoxy OEP myoglobin at 25 degrees C reversibly binds oxygen with an affinity of P50 = 0.8 mm Hg, which is similar to that of native protein. These results indicate that iron OEP serves as a prosthetic group for myoglobin with normal function, despite the significant structural and electronic difference between OEP and protoporphyrin. The unexpected functional similarity between native and OEP myoglobins was interpreted in terms of a structural perturbation at the heme distal site caused by introduction of bulky OEP into the heme pocket.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photooxidation of porphyrin in Mg-substituted horseradish peroxidase

Upon photoirradiation under aerobic conditions, the porphyrin prosthetic group in Mg-substituted horseradish peroxidase was oxidized to a mixture of its pi-cation radical and an oxidized product with an absorption band at 448 nm. The 448 nm compound was then converted to a 489 nm compound in the dark and the activation energy for the conversion was 19.3 kcal/mol. About 1 mol of O2 was consumed per mol of the 448 nm compound formed and no O2 consumption was seen in the dark reaction. The substitution of ethyl groups (meso) and hydroxyethyl groups (hemato) for the vinyl groups in protoporphyrin IX did not have an effect on the result. Under anaerobic conditions and in the presence of a suitable electron acceptor, the only photooxidation product of porphyrin was its pi-cation radical. The formation of hydroxyl radicals during irradiation under aerobic conditions was confirmed by the spin-trapping method. The formation of the above two radicals could be followed by ESR spectroscopy separately at a fixed magnetic field which was set to maximize each ESR signal. The rate of hydroxyl radical formation depended linearly on the concentration of Mg peroxidase. The photooxidation of porphyrin was slow and gave nonspecific product(s) when Mg protoporphyrin IX was present in the heme crevice of apomyoglobin or free in solution.     

Protoporphyrin IX-induced structural and functional changes in human red blood cells,haemoglobin and myoglobin

Protoporphyrin IX and its derivatives are used as photosensitizers in the photodynamic therapy of cancer. Protoporphyrin IX penetrates into human red blood cells and releases oxygen from them. This leads to a change in the morphology of the cells. Spectrophotometric studies reveal that protoporphyrin IX interacts with haemoglobin and myoglobin forming ground state complexes. For both proteins, the binding affinity constant decreases, while the possible number of binding sites increases, as the aggregation state of the porphyrin is increased. The interactions lead to conformational changes of both haemoglobin and myoglobin as observed in circular dichroism studies. Upon binding with the proteins, protoporphyrin IX releases the heme-bound oxygen from the oxyproteins, which is dependent on the stoichiometric ratios of the porphyrin: protein. The peroxidase activities of haemoglobin and myoglobin are potentiated by the protein-porphyrin complexation. Possible mechanisms underlying the relation between the porphyrin-induced structural modifications of the heme proteins and alterations in their functional properties have been discussed. The findings may have a role in establishing efficacy of therapeutic uses of porphyrins as well as in elucidating their mechanisms of action as therapeutic agents.     

The effect of tryptophanyl substitution on folding and structure of myoglobin.

Mammalian myoglobins contain two tryptophanyl residues at the invariant positions 7 (A-5) and 14 (A-12) in the N-terminal region (A helix) of the protein molecule. The crucial role of tryptophanyl residues has been investigated by site-directed mutagenesis and molecular dynamics simulation. The apomyoglobin mutants with a double W-->F substitution were found to be not correctly folded and therefore not expressed as holoprotein. The introduction of a tyrosyl residue at position 7, that is, W7YW14F, resulted in the expression of a correctly folded myoglobin. Not correctly folded apomyoglobins were found with the following mutants: W7FW14Y, W7EW14F, W7FW14E, W7KW14F, W7FW14K. Moreover, in all these cases, very low levels of expression were observed. The acid-induced denaturation curves of wild-type and folded mutant W7YW14F, obtained following the fluorescence variation of the extrinsic fluorophore 1-anilino-8-naphthalenesulfonate, revealed that the stability of the native state of mutant apoprotein is decreased, thus indicating that the replacement W-->Y in position 7 is able to restore a correct folding but not the same stability. Molecular dynamics simulation indicated that both tryptophans are involved in forming favorable, specific tertiary interactions in the native apomyoglobin structure. The lack of some of these interactions caused by tryptophanyl replacement affects the overall protein structure and may provide an explanation for the observed stability decrease. In the case of the double W-->F substitution, the simulated structure shows conclusively the domain formed by helices A, G and H to be not correctly folded. This effect is attenuated if at least one of the two residues is conserved or a tyrosyl residue replaces W7.     

EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants.

Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli. The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy. We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule. Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket. To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val. Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue.     

Anesthetic stabilization of protein intermediates: myoglobin and halothane

Halothane, an inhaled anesthetic, destabilizes the folded structure of myoglobin. To determine whether this is due to preferential interactions with less stable folded conformers of myoglobin versus the completely unfolded state, we used photoaffinity labeling, hydrogen exchange, fluorescence spectroscopy, and circular dichroism spectroscopy. Apomyoglobin was used as a model of a less stable conformer of myoglobin. Halothane destabilizes myoglobin and binds with low affinity and stoichiometry but stabilizes and binds with higher affinity to apomyoglobin. The same halothane concentration has no effect on cytochrome c stability. The apomyoglobin/halothane complex is favored at pH 6.5 as compared to pH 4.5 or pH 2.5. Halothane photoincorporates into several sites in apomyoglobin, some allosteric to the heme pocket. Guanidinium unfolding of myoglobin, monitored by CD spectroscopy, shows destabilization at less than 1.3 M Gdm but stabilization at greater than 1.3 M Gdm, consistent with the hypothesis that less stable conformers of myoglobin bind halothane preferentially. We suggest the structural feature underlying preferential binding to less stable conformers is an enlarged cavity volume distribution, since myoglobin has several intermediate-sized cavities, while cytochrome c is more well packed and has no cavities detected by GRASP. Specific binding to less stable intermediates may underlie anesthetic potentiation of protein activity.     

Fluorescence and energy transfer of tryptophans in Aplysia myoglobin.

The fluorescence decay of tryptophan residues in apo and met Aplysia limacina myoglobin and sperm whale myoglobin were measured in aqueous solution at 10 degrees-15 degrees C. In all species, multiexponential behavior was observed in which the individual components displayed unique frequency-dependent emission characteristics. The results suggest that the tryptophan fluorescence in all met samples are quenched by rapid Forster energy transfer to the heme as predicted from the crystal geometry. Fluorescence from the apo protein is similar to that in solutions of free tryptophans. In addition, the fluorescence properties of the reversible thermal denaturation of Aplysia limacina met myoglobin was investigated between 25 degrees and 75 degrees C.     

Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding.

Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Lanthanide porphyrin probes of heme proteins. Insertion of ytterbium (III) mesoporphyrin IX into apomyoglobin.

Apomyoglobin has been reconstituted with the lanthanide porphyrin complex, ytterbium(III)mesoporphyrin IX. The reconstituted material exhibits absorption and magnetic circular dichroic spectra significantly different from those of the ytterbium porphyrin itself. The sizeable, positive extrinsic Cotton effect in the Soret band of Yb-mesoporphyrin IX induced by the interactions with the globin indicates that the lanthanide porphyrin complex occupies the heme crevice.     

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.     

Biochemical properties of the heme oxygenase inhibitor, Sn-protoporphyrin. Interactions with apomyoglobin and human serum albumin

Sn-protoporphyrin is a strong competitive inhibitor of heme oxygenase and a potential pharmacological agent for the treatment of neonatal hyperbilirubinemia. Little is otherwise known about the biochemistry of tin porphyrins. We have investigated aspects of the chemistry of tin-protoporphyrin in aqueous solution and of its interactions with heme-binding proteins other than heme oxygenase, specifically apomyoglobin and human serum albumin. In the pH region 7-10, Soret region absorption studies of unbound Sn-protoporphyrin demonstrate a pH-dependent monomer-dimer equilibrium (KD congruent to 10(6) M-1 at pH 7) with little higher aggregation. Dissociation of the dimer is relatively slow at neutral pH, permitting interaction of protein ligands with monomeric and dimeric species to be distinguished and providing insights into kinetic mechanisms of porphyrin binding by heme-binding proteins. In the present study, the kinetics of interaction of Sn-protoporphyrin with apomyoglobin are presented as novel evidence that this binding proceeds by an induced fit mechanism. Binding of Sn-protoporphyrin to both apomyoglobin and serum albumin is unexpectedly weak. Between pH 7 and 9, the apparent affinity of Sn-protoporphyrin for apomyoglobin is less than 1/200 that of heme and, at pH 9, is also significantly less than that of protoporphyrin. The apparent affinity of Sn-protoporphyrin for human serum albumin is less than 1/1000 that of heme and 1/30 to 1/100 that of protoporphyrin. Competition studies between heme and Sn-protoporphyrin and between bilirubin and Sn-protoporphyrin indicate that Sn-protoporphyrin distributes differently among porphyrin-binding sites on serum albumin than does heme and that it is also not an effective competitor with bilirubin for bilirubin-binding sites. These results argue that Sn-protoporphyrin should not significantly alter normal mechanisms for the binding and transport of heme or of preformed bilirubin by serum albumin. From a more general perspective, the results indicate potentially unusual binding site selectivity by tin chelates; possible origins of this selectivity are discussed.