Biomedical subjects

T M Loehr

Publications and source records attributed to T M Loehr.

At least 19 recordsLinked to original sources

PubMed1992

Engineering of the iron site in ribonucleotide reductase to a self-hydroxylating monooxygenase.

Protein R2 of ribonucleotide reductase contains a dinuclear ferric iron center adjacent to a tyrosyl radical in the interior of the protein matrix. A patch of hydrophobic residues surrounds the iron-radical cofactor. Its importance during the oxidative generation of the iron-radical cofactor was investigated by site-directed mutagenesis of Phe-208 to tyrosine. The mutant protein R2 F208Y has prominent absorption bands at 460 and 720 nm reminiscent of those in ferric-catecholate complexes. Resonance Raman spectroscopy shows that the iron center of R2 F208Y contains a bidentate catechol ligand. The mechanism for generation of this protein-derived dihydroxyphenylalanine may be similar to the catalytic cycle of methane monooxygenase.

Base Sequence↗

PubMed1992

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.

Base Sequence↗

PubMed1991

Identification of a ferryl intermediate of Escherichia coli cytochrome d terminal oxidase by resonance Raman spectroscopy.

The 680-nm-absorbing "peroxide state" of the Escherichia coli cytochrome d terminal oxidase complex, obtained by addition of excess hydrogen peroxide to the enzyme, is shown to be a ferryl intermediate in the catalytic cycle of the enzyme. This ferryl intermediate is also created by aerobic oxidation of the fully reduced enzyme. Resonance Raman spectra with 647.1-nm excitation show an FeIV = O stretching band at 815 cm-1, a higher frequency than noted in any other ferryl-containing enzyme to date. The band shows an 16O/18O frequency shift of -46 cm-1, larger than that observed for any porphyrin ferryl species. The FeIV = O formulation was unambiguously established by oxidations of the reduced enzyme with 16O2, 18O2, and 16O18O. Only the use of a mixed-isotope gas permitted discrimination between a ferryl and a peroxo structure. A catalytic cycle for the cytochrome d terminal oxidase complex is proposed, and possible reasons for the high v(Fe = O) frequency are discussed.

Catalysis↗

PubMed1991

Resonance Raman spectra of plastocyanin and pseudoazurin: evidence for conserved cysteine ligand conformations in cupredoxins (blue copper proteins).

New resonance Raman (RR) spectra at 15 K are reported for poplar (Populus nigra) and oleander (Oleander nerium) plastocyanins and for Alcaligenes faecalis pseudoazurin. The spectra are compared with those of other blue copper proteins (cupredoxins). In all cases, nine or more vibrational modes between 330 and 460 cm-1 can be assigned to a coupling of the Cu-S(Cys) stretch with Cys ligand deformations. The fact that these vibrations occur at a relatively constant set of frequencies is testimony to the highly conserved ground-state structure of the Cu-Cys moiety. Shifts of the vibrational modes by 1-3 cm-1 upon deuterium exchange can be correlated with N-H...S hydrogen bonds from the protein backbone to the sulfur of the Cys ligand. There is marked variability in the intensities of these Cys-related vibrations, such that each class of cupredoxin has its own pattern of RR intensities. For example, plastocyanins from poplar, oleander, French bean, and spinach have their most intense feature at approximately 425 cm-1; azurins show greatest intensity at approximately 410 cm-1, stellacyanin and ascorbate oxidase at approximately 385 cm-1, and nitrite reductase at approximately 360 cm-1. These variable intensity patterns are related to differences in the electronic excited-state structures. We propose that they have a basis in the protein environment of the copper-cysteinate chromophore. A further insight into the vibrational spectra is provided by the structures of the six cupredoxins for which crystallographic refinements at high resolution are available (plastocyanins from P. nigra, O. nerium, and Enteromorpha prolifera, pseudoazurin from A. faecalis, azurin from Alcaligenes denitrificans, and cucumber basic blue protein). The average of the Cu-S(Cys) bond lengths is 2.12 +/- 0.05 A. Since the observed range of bond lengths falls within the precision of the determinations, this variation is considered insignificant. The Cys ligand dihedral angles are also highly conserved. Cu-S gamma-C beta-C alpha is always near -170 degrees and S gamma-C beta-C alpha-N near 170 degrees. As a result, the Cu-S gamma bond is coplanar with the Cys side-chain atoms and part of the polypeptide backbone. The coplanarity accounts for the extensive coupling of Cu-S stretching and Cys deformation modes as seen in the RR spectrum. The conservation of this copper-cysteinate conformation in cupredoxins may indicate a favored pathway for electron transfer.

Azurin↗

PubMed1991

Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558.

The resonance Raman spectra of neutrophil cytochrome b558 obtained upon Soret excitation indicate that the heme is low spin six-coordinate in both ferric and ferrous oxidation states; comparison with the spectra of bis-imidazole hemin suggests imidazole or imidazolate axial ligation. Minor bands attributable to vibrational motions of ring-conjugated vinyl substituents were also observed, consistent with a heme assignment of protoporphyrin IX. The spectra of deoxycholate-solubilized cytochrome b558 were indistinguishable from neutrophil plasma membranes or specific granules, as were spectra from unstimulated and phorbol myristate acetate-stimulated cells, indicating that the hemes are structurally identical in various subcellular environments and cellular physiological states. However, structural complexity was suggested by biphasic ferric-ferrous photoreduction under 413-nm illumination and the absence of an EPR spectrum for the ferric heme under conditions where simple bis-imidazole heme-containing cytochromes are expected to give detectable signals. Midpoint reduction potentials and resonance Raman spectra of the soluble cytochrome b558 from an individual with cytochrome b558 positive (type IA.2) chronic granulomatous disease were nearly identical to normal oxidase, with the exception that the deficient oxidase did not undergo heme photoreduction. Possible structural models are discussed in relation to other physical properties (ligand binding, thermodynamic potentials) exhibited by the cytochrome.

Cell Membrane↗

PubMed1990

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)

Basidiomycota↗

PubMed1990

Modelling heme d1. The spectral properties of copper(II) porphyrindiones.

The heme d1 macrocycle of Ps. aeruginosa dissimilatory nitrite reductase is an iron porphyrin-3,8-dione with a 17-acrylate substituent. We have compared the RR properties of Cu-d1, the copper(II) TME of extracted heme d1, with those of models that differ only with respect to the acrylate: Cu-17-acrylate mesoporphyrin-3,8-dione (2) and Cu-mesoporphyrin-3,8-dione (3). The RR spectrum of Cu-d1 is very similar to that of 2, including v(C = O) at approximately 1720 cm-1. Replacement of the acrylate with propionate changes the spectrum markedly. For example, the v(C = O) mode of 3 shifts to 1712 cm-1, and peaks of Cu-d1 and 2 at approximately 1400 and approximately 1535 cm-1 are shifted or absent from the spectrum of 3. FTIR spectra of 2 and 3 also differ in their voxo(C = O) frequencies. The acrylate thus has a surprisingly strong influence on the electronic structural and spectral properties of heme d1. These data provide a foundation for studies of the novel biological porphyrindione macrocycles.

Acrylates↗

PubMed1989

Comparative spectral analysis of mammalian, fungal, and bacterial catalases. Resonance Raman evidence for iron-tyrosinate coordination.

Resonance Raman spectra are reported for catalases from bovine liver, the ascomycete fungus Aspergillus niger, and the bacterium Micrococcus luteus. The vibrational frequencies of the oxidation-, spin-, and coordination number-sensitive spectral bands are indicative of high spin pentacoordinate hemes in the resting ferric enzymes of each of these organisms. This result is in accord with the crystal structure of bovine catalase (Fita, I., and Rossmann, M.G. (1985) J. Mol. Biol. 185, 21-37). In contrast, the crystallographic study of catalase from the ascomycete Penicillium vitale (Vainshtein, B. K., Melik-Adamyan, W. R., Barynin, V. V., Vagin, A.A., Grebenko, A. I., Borisov, V. V., Bartels, K. S., Fita, I., and Rossmann, M. G. (1986) J. Mol. Biol. 188, 49-61) showed electron density on the distal side of the heme which could imply the presence of a sixth ligand, possibly a water molecule. However, both of these crystallographic studies showed the proximal ligand in catalase to be a tyrosine. The present study confirms tyrosinate coordination in each of the three catalases from the appearance of selected resonance-enhanced tyrosine vibrational modes. The most characteristic band is the tyrosinate ring mode at approximately 1612 cm-1 which is maximally enhanced with 488.0 nm excitation. The appearance of tyrosinate modes at 1607 and 1245 cm-1 in the resonance Raman spectra of M. luteus cyano catalase serves to identify tyrosine as an axial ligand in bacterial as well as eukaryotic catalases. Unlike non-heme iron tyrosinate proteins, whose resonance Raman spectra are dominated by several intense bands diagnostic of tyrosine ligation, the heme-linked tyrosine modes are not easily distinguished from the large number of porphyrin vibrations.

Animals↗

PubMed1989

Spectroscopic analysis of chlorophyll model complexes: methyl ester ClFe(III)pheophorbides.

As models for chlorophyll a (Chl a), methyl ester ClFe(III)pheophorbides (1, pheophorbide a; 2, mesopheophorbide a; and 3, mesopyropheophorbide a) were examined by Fourier transform infrared (FTIR) absorption and resonance Raman (RR) spectroscopy. The infrared (IR) chlorin band above 1600 cm-1, assigned as a Ca-Cm mode (Andersson et al. (1987) J. Am. Chem. Soc. 109, 2908-2916) is shown to be metal-sensitive and responsive to spin state and coordination number for dihydroporphyrins, as well as being diagnostic for the chlorin vs. porphyrin or bacteriochlorin macrocycle. Frequency variations for this metallochlorin IR band thus parallel those of the v10 RR mode of porphyrins in their predictive utility. Qy excitation SERRS spectra of Chl a were compared with Qy excitation RR spectra of 1 and methyl Ni(II)pyropheophorbide a. The data demonstrate that 5-coordinate ClFe(III)pheophorbides are better models for chlorophylls than are ruffled 4-coordinate Ni(II)pheophorbides. Major spectral differences between the three chlorophyll models are associated with the C-9 keto and/or C-10 carbomethoxy vibrational modes. The approx. 1700 cm-1 IR band was formerly assigned solely to v(C = O) of the C-9 keto group. However, this IR feature shifts down to approx. 1685 cm-1 and nearly doubles in intensity when the C-10 carbomethoxy is removed, as for 3. Similar frequency downshifts coupled with intensity increases in the IR are found in the literature on chlorophylls. RR spectra of pheophorbides having the C-10 carbomethoxy group (1 and 2) have bands at both approx. 1700 and approx. 1735 cm-1. However, the C-9 keto v(C = O) mode of pyrophorbins also downshifts to approx. 1685 cm-1, as in the IR spectra. The approx. 1735 cm-1 ester RR mode disappears in the case of pyrophorbins, and is never RR active for nonconjugated esters of porphyrins or chlorins. These data demonstrate an interaction between the C-10 and C-9 carbonyls of phorbins. They also indicate that phorbins tend toward conjugation of the C-10 ester. Biological examples of such conjugation effects have recently been reported, e.g., for the Chl a pi-cation radical (Heald et al. (1988) J. Phys. Chem. 92, 4820-4824). Because the phorbin E ring is the major structural feature distinguishing chlorophylls from non-photosynthetic systems, the participation of the C-10 ester in ring conjugation is suggestive of its biological importance.

Chlorophyll↗

PubMed1989

Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center.

Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety. When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1. Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH [Sjöberg, B. M., Loehr, T. M., & Sanders-Loehr, J. (1987) Biochemistry 26, 4242], the present studies indicate that the intensity of this mode is dependent primarily on input laser power. The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode. This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2. One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate.

Escherichia coli↗

PubMed1988

Spectral characterization of manganese peroxidase, an extracellular heme enzyme from the lignin-degrading basidiomycete, Phanerochaete chrysosporium.

Manganese peroxidase (MnP) is a component of the lignin degradation system of the basidiomycetous fungus, Phanerochaete chrysosporium. This novel MnII-dependent extracellular enzyme (Mr = 46,000) contains a single protoporphyrin IX prosthetic group and oxidizes phenolic lignin model compounds as well as a variety of other substrates. To elucidate the heme environment of this enzyme, we have studied its electron paramagnetic resonance and resonance Raman spectroscopic properties. These studies indicate that the native enzyme is predominantly in the high-spin ferric form and has a histidine as fifth ligand. The reduced enzyme has a high-spin, pentacoordinate ferrous heme. Fluoride and cyanide readily bind to the sixth coordination position of the heme iron in the native form, thereby changing MnP into a typical high-spin, hexacoordinate fluoro adduct or a low-spin, hexacoordinate cyano adduct, respectively. EPR spectra of 14NO- and 15NO-adducts of ferrous MnP were compared with those of horseradish peroxidase (HRP); the presence of a proximal histidine ligand was confirmed from the pattern of superhyperfine splittings of the NO signals centered at g approximately equal to 2.005. The appearance of the FeII-His stretch at approximately 240 cm-1 and its apparent lack of deuterium sensitivity suggest that the N delta proton of the proximal histidine of the enzyme is more strongly hydrogen bonded than that of oxygen carrier globins and that this imidazole ligand may be described as having a comparatively strong anionic character. Although resonance Raman frequencies for the spin- and coordination-state marker bands of native MnP, nu 3 (1487), nu 19 (1565), and nu 10 (1622 cm-1), do not fall into frequency regions expected for typical penta- or hexacoordinate high-spin ferric heme complexes, ligation of fluoride produces frequency shifts of these bands very similar to those observed for cytochrome c peroxidase and HRP. Hence, these data strongly suggest that the iron in native MnP is predominantly high-spin pentacoordinate. Analysis of the Raman frequencies indicates that the dx2-y2 orbital of the native enzyme is at higher energy than that of metmyoglobin. These features of the heme in MnP must be favorable for the peroxidase catalytic mechanism involving oxidation of the heme iron to FeIV. Consequently, it is most likely that the heme environment of MnP resembles those of HRP, cytochrome c peroxidase, and lignin peroxidase.

Basidiomycota↗

PubMed1988

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans.

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.

Azurin↗

PubMed1988

Nitric oxide adducts of the binuclear iron site of hemerythrin: spectroscopy and reactivity.

Nitric oxide forms adducts with the binuclear iron site of hemerythrin (Hr) at [Fe(II),Fe(II)]deoxy and [Fe(II),Fe(III)]semimet oxidation levels. With deoxyHr our results establish that (i) NO binds reversibly, forming a complex which we label deoxyHrNO, (ii) NO forms a similar but distinct complex in the presence of fluoride, which we label deoxyHrFNO, (iii) NO is directly coordinated to one iron atom of the binuclear pair in these adducts, most likely in a bent end-on fashion, and (iv) the iron atoms in the binuclear sites of both deoxyHrNO and deoxyHrFNO are antiferromagnetically coupled, thereby generating unique electron paramagnetic resonance (EPR) detectable species. The novel EPR signal of deoxyHrNO (deoxyHrFNO) with g[[ = 2.77 (2.58) and g = 1.84 (1.80) is explained by the magnetic interaction of the Fe(II) (S' = 2) and [FeNO]7 (S = 3/2) centers observed by Mössbauer spectroscopy. Antiferromagnetic coupling leads to a ground state of Seff = 1/2. Analysis of the EPR parameters using the isotropic spin-exchange Hamiltonian, Hex = 2JS3/2.S2, and including zero-field splitting leads to a coupling constant, -J approximately 23 cm-1, for deoxyHrNO. The resonance Raman spectrum of deoxyHrNO shows features at 433 and 421 cm-1 that shift downward with 15N16O and that are assigned to stretching and bending modes, respectively, of the [FeNO]7 unit. Sensitivity of the bending mode to D2O suggests that bound NO participates in hydrogen bonding. We propose that the terminal oxygen atom of NO is hydrogen bonded to the proton of the mu-hydroxo bridge in the Fe-(OH)-Fe unit. A bent Fe-N-O geometry is supported by spectroscopic and structural comparisons to synthetic complexes and is consistent with a limiting [FeII,FeIIINO-] formulation for deoxyHrNO. Reversibility of NO binding to deoxyHr is demonstrated by bleaching of the optical and EPR spectra of deoxyHrNO upon additions of excess N3- or CNO-. DeoxyHrNO undergoes autoxidation under anaerobic conditions over the course of several hours. The product of this autoxidation appears to be an EPR-silent NO adduct of semimetHr. The formal one-electron oxidations of the binuclear iron site of deoxyHr by NO and by HNO2 can conceivably occur with no net change in charge on the iron site. In contrast, autoxidation of oxy- to metHr requires a change in net charge on the iron site, which may provide a kinetic barrier.

Animals↗

PubMed1987

Hydrogen bonding of sulfur ligands in blue copper and iron-sulfur proteins: detection by resonance Raman spectroscopy.

The resonance Raman spectrum of the blue copper protein azurin from Alcaligenes denitrificans exhibits nine vibrational modes between 330 and 460 cm-1, seven of which shift 0.4-3.0 cm-1 to lower energy after incubation of the protein in D2O. These deuterium-dependent shifts have been previously ascribed to exchangeable protons on imidazole ligands [Nestor, L., Larrabee, J. A., Woolery, G., Reinhammar, B., & Spiro, T. G. (1984) Biochemistry 23, 1084] or to exchangeable protons on amide groups which are hydrogen bonded to the cysteine thiolate ligands (a feature common to all blue copper proteins of known structure). In order to distinguish between these two possibilities, a systematic investigation of Fe2S2(Cys)4-containing proteins was undertaken. Extensive hydrogen bonding between sulfur ligands and the polypeptide backbone had been observed in the crystal structure of ferredoxin from Spirulina platensis. The resonance Raman spectrum of this protein is typical of a chloroplast-type ferredoxin and exhibits deuterium-dependent shifts of -0.3 to -0.5 cm-1 in the Fe-S modes at 283, 367, and 394 cm-1 (assigned to the bridging sulfurs) and -0.6 to -0.8 cm-1 in the Fe-S modes at 328 and 341 cm-1 (assigned to the terminal cysteine thiolates). Considerably greater deuterium sensitivity is observed in the Raman spectra of spinach ferredoxin and bovine adrenodoxin, particularly for the symmetric stretching vibration of the Fe2S2 moiety at approximately 390 cm-1. This feature decreases by 0.8 and 1.1 cm-1, respectively, for the two oxidized proteins in D2O and by 1.8 cm-1 for reduced adrenodoxin in D2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

PubMed1987

Identification of a hydroxide ligand at the iron center of ribonucleotide reductase by resonance Raman spectroscopy.

The resonance Raman spectrum of protein B2 of ribonucleotide reductase from Escherichia coli shows several features to its oxo-bridged binuclear iron center. A peak at 492 cm-1 is assigned to the symmetric stretch of the Fe-O-Fe moiety on the basis of its 13-cm-1 shift to lower energy upon 18O substitution. The 18O species shows an additional peak at 731 cm-1, which is a good candidate for the asymmetric stretch of the Fe-O-Fe moiety. Its exact location in the 16O species is obscured by the presence of a protein tryptophan vibration at 758 cm-1. A third resonance-enhanced peak at 598 cm-1 is identified as an Fe-OH vibration on the basis of its 24-cm-1 shift to lower energy in H2 18O, its 2-cm-1 shift to lower energy in D2O, and its pH-dependent intensity. A hydrogen-bonded mu-oxo bridge similar to that in hemerythrin is suggested by the unusually low frequency for the Fe-O-Fe symmetric stretch and the 3-cm-1 shift to higher energy of vs(Fe-O-Fe) in D2O. From the oxygen isotope dependence of vs(Fe-O-Fe), an Fe-O-Fe angle of 138 degrees can be calculated. This small angle suggests that the iron center consists of a tribridged core as in hemerythrin. A model for the binuclear iron center of ribonucleotide reductase is presented in which the hydroxide ligand sites provide an explanation for the half-of-sites reactivity of the enzyme.

Escherichia coli↗

PubMed1987

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme.

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)

Basidiomycota↗

PubMed1987

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Alcaligenes↗

PubMed1985

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme.

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.

Basidiomycota↗