Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

Resonance Raman characterization of the heme prosthetic group in eosinophil peroxidase

The resonance-enhanced Raman spectrum of eosinophil peroxidase (EPO) from horse and human eosinophils is reported. Based upon the spectral energies, distribution and depolarization ratios of the high-frequency skeletal modes and upon the presence of weak bands assignable to vinyl substituent groups, we conclude that the heme prosthetic group is high-spin, 6 coordinate protoporphyrin. The Raman spectrum reveals clear differences from lactoperoxidase (LPO), an enzyme which appears nearly structurally isomorphous by other physical techniques; the data indicate a stronger axial 6th ligand in EPO. Mechanistic implications are discussed in relation to LPO and myeloperoxidase, an enzyme present in neutrophils and monocytes which contains a unique functional active-site chlorin.     

Resonance Raman evidence for an exchangeable protein hydrogen associated with the heme a group of cytochrome oxidase

When cytochrome-c oxidase is soaked in D2O, downshifts of the cytochrome a formyl C = O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D2O-induced RR changes are fully developed during 3-5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H-bonds to the heme formyl. The rate of the D2O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H-bond is directly involved in the redox-linked mechanism of proton pumping.     

Resonance Raman evidence for substrate reorginization in the active site of papain.

Resonance Raman spectra were obtained for the acylenzyme 4-dimethylamino-3-nitro(alpha-benzamido)cinnamoyl-papain prepared using the chromophoric substrate methyl 4-dimethylamino-3-nitro(alpha-benzamido)cinnamate. These spectra contained vibrational spectral data of the acyl residue while covalently attached to the active site and could be used to follow directly acylation and deacylation kinetics. Spectra were obtained at pH values ranging from those where the acyl-enzyme is relatively stable (pH 3.0, tau 1/2 congruent to 800 s) to those where it is relatively unstable (pH 9.2, tau 1/2 congruent to 223 s). Throughout this range acyl-enzyme spectra differed completely from that of the free substrate or the product (4-dimethylamino-3-nitro(alpha-benzamido)cinnamic acid) indicating that a structural change occurred on combination with the active site. The spectra are consistent with rearrangement of the alpha-benzamido group in the bound substrate, -NH--C(==O)Ph becoming --N==C(--OX)Ph, where the bonding to oxygen is unknown. Superimposed on these large differences, small changes in acyl-enzyme spectra also occurred as pH was raised to decrease the half-life. All of the above spectral perturbations are consistent with a structural change in the acyl-enzyme which precedes the rate-determining step in deacylation. Thus, deacylation proceeds from an acyl residue structure differing from that of the substrate in solution. Upon acid denaturation the spectrum characteristic of the intermediate reverts to one closely resembling the substrate, demonstrating that a functioning active site is necessary to produce the observed differences. Spectra in D2O of native acyl-enzyme were identical with those in H2O, indicating that the observed differences in rate constant were not due to solvent-induced structural changes. Activated papain purified by crystallization or by affinity chromatography formed the acyl-enzyme. However, the kinetics of formation and deacylation differed between these materials, as did the spectral properties. Small differences in active-site structure are considered to be responsible for this effect, and it is suggested that such spectral perturbations may be useful in directly relating small differences in structure of the substrate in the active site with corresponding differences in kinetics.     

Two gel states of cerebrosides. Calorimetric and Raman spectroscopic evidence.

The enthalpy of the gel-to-liquid crystalline phase transition for kerasin (15.8 kcal/mol) is found to be markedly higher than that for phrenosin and unfractionated bovine brain cerebrosides (about 7 kcal/mol). Evidence for a higher degree of order in the hydrocarbon chains and a different configuration in the polar region of kerasin is supplied by Raman spectroscopic parameters for these gel phases. The high transition enthalpy for kerasin is ascribed to a lesser accommodation of gauche conformers in the hydrocarbon chains just below the transition temperature. The thermodynamic behavior of these cerebroside fractions, including hysteresis in kerasin gels, is compared to that previously reported for sphingomyelins.     

Resonance Raman investigation of an enzyme-inhibitor complex.

The resonance Raman spectrum has been recorded for two different binary complexes formed between 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (zincon) and liver alcohol dehydrogenase. The shifts in the zincon spectrum upon complexation with enzyme in one complex are similar to those in model compounds containing azo or formazyl linkages upon complexation of these with zinc. The results are interpreted in terms of complexation of zincon to a zinc atom at the enzyme active site. Since zincon is a coenzyme competitive inhibitor, it is probably bound at or near the coenzyme binding site; the results of this study, therefore, are useful in understanding the chemistry of zinc at the enzyme active site.     

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 ¹⁶O₂ and ¹⁸O₂ 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.     

Resonance Raman spectroscopic characterization of compound III of lignin peroxidase

Resonance Raman (RR) spectra of several compounds III of lignin peroxidase (LiP) have been measured at 90 K with Soret and visible excitation wavelengths. The samples include LiPIIIa (or oxyLiP) prepared by oxygenation of the ferrous enzyme, LiPIIIb generated by reaction of the native ferric enzyme with superoxide, LiPIIIc prepared from native LiP plus H2O2 followed by removal of excess peroxide with catalase, and LiPIII* made by addition of excess H2O2 to the native enzyme. The RR spectra of these four products appear to be similar and, thus, indicate that the environments of these hexacoordinate, low-spin ferriheme species must also be very similar. Nonetheless, the Soret absorption band of LiPIII* is red-shifted by 5 nm from the 414-nm maximum common to LiPIIIa, -b, and -c [Wariishi, H., & Gold, M.H. (1990) J. Biol. Chem. 265, 2070-2077]. Analysis of the iron-porphyrin vibrational frequencies indicates that the electronic structures for the various compounds III are consistent with an FeIIIO2.-formulation. The spectral changes observed between the oxygenated complex and the ferrous heme of lignin peroxidase are similar to those between oxymyoglobin and deoxymyoglobin. The contraction in the core sizes in compound III relative to the native peroxidase is analyzed and compared with that of other heme systems. EPR spectra confirm that the high-spin ferric form of the native enzyme, with an apparent g = 5.83, is converted into the EPR-silent LiPIII* upon addition of excess H2O2. Its magnetic behavior may be explained by anti-ferromagnetic coupling between the low-spin FeIII and the superoxide ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman spectroscopic evidence that carp deoxyhemoglobin remains in a T-like quaternary structure at high pH: implications for cooperativity

Resonance Raman spectroscopy shows the Fe-proximal imidazole stretching band to shift from 215 to 219 cm-1 between human deoxyhemoglobin (deoxy-Hb) and a Hb sample which is 75% oxygenated, demonstrating that the T-R quaternary structure switch can be monitored by resonance Raman spectroscopy in native Hb at equilibrium. For deoxy-Hb from carp, the band is at 215 cm-1 at pH 9 as well as pH 6, contrary to previous reports of an elevated frequency at high pH. The invariance of this frequency over a large affinity difference is in contrast to a recent report of continuously varying vFe-ImH frequencies for human mutant deoxy-Hb's. The band shifts to 219 cm-1 for carp Hb at pH 9 when O2 is bound to only 20% of the hemes. The spectra are consistent with a T-R switch upon binding approximately 0.5 O2 per Hb, on the average, although the number may be higher if the binding affinity is higher for alpha- than for beta-chains. The 0.5 value, in conjunction with the weak cooperativity observed for carp Hb at pH 9, is incompatible with a value of the allosteric constant, L = (T0)/(R0), large enough to prevent the vFe-ImH band from shifting detectably at pH 9 in the absence of O2. The possibility of functionally important intermediate structures is discussed.     

Resonance Raman evidence for the activation of dioxygen in horseradish oxyperoxidase

Resonance Raman spectroscopy has been employed to investigate the molecular bases for the markedly different properties of horseradish oxyperoxidase and oxymyoglobin. The porphyrin core of oxyperoxidase is slightly more expanded with the iron atom closer to the porphyrin plane, and there is greater iron d pi-to-oxygen pi backbonding compared to oxymyoglobin. The iron-oxygen (stretching or bending) bands are observed at 570 and 562 cm-1, respectively, for oxymyoglobin and oxyperoxidase, and the iron-His stretching bands have been tentatively identified at 276 and 289 cm-1, respectively. It is suggested that the stronger iron-His bond in oxyperoxidase facilitates greater iron d pi-to-oxygen pi backdonation by raising the energy of the iron d pi orbitals closer to the energy of the oxygen pi orbitals. This weakens the O-O bond and activates dioxygen for use as an electron acceptor in the peroxidase-oxidase reaction.     

Resonance Raman evidence for an unusually strong exogenous ligand-metal bond in a monomeric nitrosyl manganese hemoglobin

Resonance Raman spectroscopy has been employed to determine the vibrational modes of monomeric nitrosyl manganese Chironomus thummi thummi hemoglobin (CTT IV). This insect hemoglobin has no distal histidine. By applying various isotope-labeled nitric oxides (14N16O, 15N16O, 14N18O), we have identified the Mn11-NO stretching model at 628 cm-1, the Mn11-N-O bending mode at 574 cm-1 and the N-O stretching mode at 1735 cm-1. The results suggest a strong Mn11-NO bond and a weak N-O bond. The vinyl group substitution does not influence the nu (Mn11-NO), delta (Mn11-N-O) and nu (N-O) vibrations. The Mn11-NO stretching frequency is insensitive to distal histidine interactions with NO, whereas the N-O stretching frequency is sensitive. Nitric oxide also binds to Met manganese CTT IV to form an Mn111. NO complex which undergoes a slow but complete autoreduction resulting in the Mn11.NO species. In manganese meso-IX CTT IV, the Mn111. NO Mn11. NO conversion alters the intensities of the porphyrin ring modes at 342, 360, 1587 and 1598 cm-1, but shifts the frequencies at 1504 and 1633 cm-1 (in Mn111.NO) to 1497 and 1630 cm-1 (in Mn11. NO), respectively. The unshifted marker line at 1378 cm-1 reflects the fact that the pi electron densities of the porphyrin ring are the same in the two complexes.     

Raman spectroscopic evidence for a disulfide bridge in calf gamma II crystallin

Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical gamma II crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of gamma II as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-1), the native form shows a line near 511 cm-1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-1. In every other spectral region, both native and reduced gamma II forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.     

Raman spectroscopic evidence for structural changes in poly-L-lysine induced by an approximately 50 mT static magnetic field

We have explored the mechanism of coupling of an approximately 50 mT static magnetic field with the α helices of poly-L-lysine. Structural changes in poly-L-lysine were determined by Raman spectroscopy. Our testable hypothesis is that static magnetic fields of this magnitude can couple with the α-helical segments of the polypeptide, and, as a result, the structure of the polypeptide is significantly altered. Our model further suggests that a static magnetic field can promote protein unfolding and can prevent refolding. © 1996 Wiley-Liss, Inc.     

Resonance Raman spectroscopic study of the tryptic 39-kDa fragment of phytochrome

The 39-kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state P(r) reveals far-reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the P(r) phototransformation, denoted as P(bl), is more closely related to that of the P(fr) precursor of the native phytochrome, i.e. meta-R(C), rather than to that of P(fr) itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in P(bl) so that, unlike in P(fr), the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of P(fr), cannot be established in the 39-kDa fragment due to the lack of the major C-terminal part of the protein. These findings, furthermore, support the view that the meta-R(C)-->P(fr) transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome.     

Resonance Raman scattering on the haem group of cytochrome c

Resonance Raman spectra of the haem group of 8 × 10^?5 M horse heart ferro- and ferricytochrome c solutions have been obtained. The spectra are almost identical to that of haemoglobin. The frequency of the Raman line near 1370 cm^?1, which in haemoglobin is sensitive to the position of the haem iron, indicates that the iron atom of cytochrome c lies in the plane of the porphyrin for both oxidation states.     

Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase

Resonance Raman data are reported for the redox-activated form of galactose oxidase from Dactylium dendroides. Excitation within the red (659 nm) and blue (457.9 nm) absorption bands leads to strong resonance enhancement of ligated tyrosine vibrational modes at 550, 1170, 1247, 1484, and 1595 cm-1. The ring mode frequencies are unusually low, indicating a decreased bond order in the ring. The spectra clearly differ in both frequencies and relative intensities from those characteristic of known aromatic pi-radicals. Enhancement of tyrosine ring modes on excitation within absorption bands previously associated with the presence of the radical in the active site suggests that the ligated tyrosine residue is present in the radical site and may stabilize this radical species through formation of a charge transfer complex. A dramatically different Raman spectrum is observed for the N3- adduct of galactose oxidase, exhibiting a single strong 1483 cm-1 feature. The intense visible-near IR absorption bands for galactose oxidase may derive from transitions within a charge transfer complex between an aromatic free radical and a tyrosine-copper complex.