Ultraviolet resonance Raman examination of horse apomyoglobin acid unfolding intermediates.

We have used UV resonance Raman spectroscopy to study the acid-induced denaturation of horse apomyoglobin (apoMb) between pH 7. 0 and 1.8. The 206.5 nm excited Raman spectra are dominated by amide vibrations, which are used to quantitatively determine the apoMb secondary structure. The 229 nm excited Raman spectra are dominated by the Tyr and Trp Raman bands, which are analyzed to examine changes of Tyr and Trp environments and solvent exposures. We observe two partially unfolded apoMb intermediates at pH 4 and pH 2, while we observe only one partially unfolded holoMb intermediate at 2, in which the G and H helices are mainly intact, while the rest of protein is unfolded. This partially unfolded holoMb intermediate at pH 2 is essentially identical to the pH 2 apoMb intermediate. The partially unfolded pH 4 apoMb intermediate is composed of the three folded A, G, and H helices and contains 38% helical structure. The changes in the Trp Raman cross sections during the acid-induced denaturation indicates that Trp 7 is likely to be fully exposed in the apoMb pH 4 intermediate and that the A helix melts with a pKa approximately 3.5.     

Implications for an ionized alkyl-enzyme intermediate during StEH1-catalyzed trans-stilbene oxide hydrolysis

The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.     

Resonance raman studies of a c type algal cytochrome. Deuterium shifts and a comparison with mammalian cytochrome c.

A c type cytochrome isolated from Synechococcus lividus grown on water and 2H2O media, has been studied by resonance Raman spectroscopy. The spectra were taken on the oxidized and reduced protein with excitation within the Soret band at 441.6 nm to determine whether individual resonance Raman bands of the heme shift upon deuterium substitution and also to provide a comparison with the spectra of horse heart cytochrome c. Some of the shifts observed with the deuterated heme c are larger than the corresponding shifts in meso-deuterated metalloporphyrins suggesting mixing of peripheral substituent vibrations with the skeletal modes of the porphyrin macrocycle. The algal cytochrome exhibits resonance Raman spectra roughly similar to those of horse heart cytochrome c, consistent with its optical absorption spectra which is typical of c type cytochromes, although a detailed comparison reveals note-worthy differences between the spectra of the two proteins; this may be a reflection of the effect of non-methionine ligands and protein environment on the vibrations of the c type heme in the algal cytochrome.     

Identification and properties of an oxoferryl structure in myeloperoxidase compound II

Myeloperoxidase compound II has been characterized by using optical absorption and resonance Raman spectroscopies. Compared to compounds II in other peroxidases, the electronic and vibrational properties of this intermediate are strongly perturbed due to the unusual active-site iron chromophore that occurs in myeloperoxidase. Despite this difference in prosthetic group, however, other properties of myeloperoxidase compound II are similar to those observed for this intermediate in the more common peroxidases (horseradish peroxidase in particular). Two forms of the myeloperoxidase intermediate species, each with distinct absorption spectra, are recognized as a function of pH. We present evidence consistent with interconversion of these two forms via a heme-linked ionization of a distal amino acid residue with a pKa congruent to 9. From resonance Raman studies of isotopically labeled species at pH 10.7, we identify an iron-oxygen stretching frequency at 782 cm-1, indicating the presence of an oxoferryl (O = FeIV) group in myeloperoxidase compound II. We further conclude that the oxo ligand is not hydrogen bonded above the pKa but possibly exhibits oxygen exchange with the medium at pH values below the pKa due to hydrogen bonding of the oxo ligand to the distal protein group.     

Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. I. Spectroscopic and physico-chemical properties of hematoxylin and hematein.

Analytically pure hematoxylin (Htx), pentaacetylhematoxylin (PAHtx), and hematein (Hm) were isolated and characterized by 1H-NMR spectroscopy. The VIS/UV spectra of Htx and Hm were recorded in MeOH and in H2O at various pH values. The molar extinction coefficients of the long wavelength absorption bands are reported. The pKa value for the 1st acidic dissociation step of Hm has been determined from the pH dependency of the absorption spectra of Hm in aqueous buffer solutions. Finally, the absorption spectra are qualitatively discussed.     

Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.

Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.     

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.     

Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.

Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.     

Ribonucleotide reductase: A structural study of the dimeric iron site

The iron-containing B2 subunit of ribonucleotide reductase from Escherichia coli has been investigated by Raman spectroscopy. Both the tyrosyl radical-containing native protein and the radical-free protein exhibit a resonance-enhanced Raman band at 500 cm^?1. This band is assigned to an Fe-O vibrational mode arising from an oxygen-containing ligand. The failure to observe any tyrosinate ring modes makes it unlikely that ribonucleotide reductase is an iron-tyrosinate protein and rules out tyrosinate oxygen as a ligand. It is proposed that the 500 cm^?1 band in ribonucleotide reductase is analogous to the 510 cm^?1 Fe-O vibrational mode of methemerythrin and arises from an oxo- or carboxylate-bridge between the antiferromagnetically-coupled Fe(III) ions.     

Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.

Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

Proteins of the thermophilic fungus Humicola lanuginosa. II. Some physicochemical properties of a cytochrome c

A cytochrome c from Humicola lanuginosa is unique among eukaryotic cytochromes c in having phenylalanine as Residue 74. This protein has certain properties which differ from those of other cytochromes c to which it is generally similar. The Humicola cytochrome c is as stable as horse heart cytochrome c in urea, but more stable than both horse heart and yeast cytochromes c in acidic and alkaline conditions. Spectrophotometric titration of the four tyrosyl residues of the Humicola protein was nonsigmoidal with a pK_app of 11.4. Solvent perturbation difference spectra indicate that 50% of the tyrosyl residues are exposed to solvent in the native protein, and that the single tryptophanyl and all four tyrosyl residues become exposed in 8 m urea. Certain unusual features in both the optical rotatory dispersion and circular dichroism spectra in the 290-250-nm region are tentatively attributed to the substitution of phenylalanine for tyrosine at position 74.     

Conformational Change of Chymotryptic Myosin Rod

Myosin rod was prepared from hen myosin by chymotryptic digestion. The indigested myosin was successfully removed by ultracentrifugation following alcohol treatment. No significant difference in UV absorption and CD spectra was observed between pH 7.0 and pH 10.5 for both myosin rod and myosin. When pH was raised to 11.7, the phenolic groups of the tyrosyl residues were ionized, and the helical configuration of the myosin rod and myosin could not withstand the electrostatic repulsion. When pH was further raised to 13.6, “abnormal” tyrosyl residues were ionized, resulting in decreased helix content. However, the myosin rod was stabler and less flexible against pH change than myosin, because of the lower content of tyrosyl residues in myosin rod.     

A model for the misfolded bis-His intermediate of cytochrome c: the 1-56 N-fragment

We have characterized the ferric and ferrous forms of the heme-containing (1-56 residues) N-fragment of horse heart cytochrome c (cyt c) at different pH values and low ionic strength by UV-visible absorption and resonance Raman (RR) scattering. The results are compared with native cyt c in the same experimental conditions as this may provide a deeper insight into the cyt c unfolding-folding process. Folding of cyt c leads to a state having the heme iron coordinated to a histidine (His18) and a methionine (Met80) as axial ligands. At neutral pH the N-fragment (which lacks Met80) shows absorption and RR spectra that are consistent with the presence of a bis-His low spin heme, like several non-native forms of the parental protein. In particular, the optical spectra are identical to those of cyt c in the presence of a high concentration of denaturants; this renders the N-fragment a suitable model to study the heme pocket microenvironment of the misfolded (His-His) intermediate formed during folding of cyt c. Acid pH affects the ligation state in both cyt c and the N-fragment. Data obtained as a function of pH allow a correlation between the structural properties in the heme pocket of the N-fragment and those of non-native forms of cyt c. The results underline that the (57-104 residues) segment under native-like conditions imparts structural stability to the protein by impeding solvent access into the heme pocket.     

Lactoperoxidase-catalyzed iodination of horse cytochrome c:monoiodotyrosyl 74 cytochrome c.

Iodination of horse cytochrome c with the lactoperoxidase-hydrogen peroxide-iodide system results initially in the formation of the monoiodotyrosyl 74 derivative. This singly modified protein was obtained in pure form by ion exchange chromatography and preparative column electrophoresis. It shows an intact 695 nm absorption band, the midpoint potential of the native protein, a nuclear magnetic resonance spectrum which indicates an undisturbed heme crevice structure, a normal reaction with antibodies directed against native horse cytochrome c, and circular dichroic spectra in which the only changes from those of the native protein can be ascribed to the spectral properties of iodotyrosine itself. This conformationally intact derivative reacts with the succinate-cytochrome c reductase and the cytochrome c oxidase systems of beef mitochondrial particle preparations indistinguishably from the unmodified protein, showing that the region including tyrosine 74 is not involved in these enzymic electron transfer functions of the protein. The circular dichroic spectra of this derivative indicate that the minima observed at 288 and 282 nm in the spectrum of native ferricytochrome c originate from tyrosyl residue 74.     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Resolution of independently titrating spectral components in the ultraviolet circular dichroism of subtilisin enzymes by matrix rank analysis.

The ultraviolet circular dichroism of di-isopropylphophoryl-subtilisins Carlsberg and Novo (EC 3.4.21.14) has been examined as a function of pH. The CD of these enzymes below 260 nm is invariant over the pH interval 4 to 12, below or above which spectral changes occur suggesting a transition to a random coil form. Above pH 8 contributions due to the ionization of tyrosyl residues appear in the CD above 260 nm as bands shifted to longer wavelengths. Three independently titratable components, obtained by matrix rank analysis, account for the observed CD spectral changes above 260 nm of Dip-subtilisin Carlsberg in the pH interval 8 to 12. By contrast, two components were derived for the Novo enzyme. The identities of the matrix rank components were surmised from their apparent pKa values. One component of both subtilisin enzymes corresponds to the CD of the "buried" or irreversibly titratable tyrosyl residues of the enzyme. The other matrix rank components correspond to the CD of the "exposed" or freely ionizable tyrosyl residues. These residues are optically active only in the ionized state. Two types of "exposed" tyrosyl residues, arising because of differing sensitivity to the ionization of the "partially buried" or abnormally titrating tyrosyl residues, are evident in Dip-subtilisin Carlsberg. A pH-induced local conformational change in this enzyme is proposed to account for this behavior. The "partially buried" tyrosyl residues of both subtilisins appear to be devoid of optical activity in either the tyrosyl or tyrosylate form.     

Measurement of the individual pKa values of acidic residues of hen and turkey lysozymes by two-dimensional 1H NMR.

The pH dependence of the two-dimensional 1H nuclear magnetic resonance spectra of hen and turkey egg-white lysozymes has been recorded over the pH range 1-7. By monitoring the chemical shifts of the resonances of the various protons of ionizable residues, individual pKa values for the acidic residues have been determined for both proteins. The pKa values are displaced, with the exception of those of the residues in the active site cleft, by an average of 1 unit to low pH compared to model compounds.     

Proton magnetic resonance spectra of adrenodoxin: features of the aromatic region

This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)