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The spectroscopic and mechanistic properties of the Cu-containing active site of azurin from Pseudomonas aeruginosa were investigated by the construction of a mutant in which one of the ligands of the metal, His46, was replaced by a glycine. Although the mutation creates a hole in the interior of the protein, the 3D structure of the protein does not change to any appreciable extent. However, the spectroscopic (optical, resonance Raman, EPR) properties of the mutant protein are strongly affected by the mutation. In the presence of external ligands, the properties of the original wild-type protein are restored to a smaller or larger extent, depending on the ligand. It is concluded that the hole created by the mutation, even though it is completely buried inside the protein, can be filled by external ligands, often resulting in the creation of a mixture of so-called type-1 and type-2 copper sites. Also, the redox properties (midpoint potential, kinetics of reduction/oxidation) appeared to be strongly affected by the mutation and the presence of external ligands. The results are compared with previous results obtained on the mutant His117Gly.

Quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni (QH-EDH) contains two cofactors, 2,7,9-tricarboxy-1H-pyrrolo[2,3-f]quinoline-4,5-dione (PQQ) and heme c. Since previous studies on the kinetics of this enzyme suggested that both participate in electron transfer, spectroscopic investigations were performed of the oxidized and reduced holo- and apoenzyme (without PQQ but with heme c) to reveal the nature of the interaction between the two redox centers. From this it appears that the properties of the heme in the enzyme are affected by the presence of PQQ, as judged from the shift of the maxima in the ultraviolet/visible absorption spectra of the heme moiety in both reduced and oxidized QH-EDH and the 60-mV increase of the heme midpoint redox potential caused by PQQ addition. Also 1H-NMR spectroscopy was indicative for interaction since binding of PQQ induced shifts in the resonances of the methyl groups of the porphyrin ring in the oxidized form of the apoenzyme and a shift in the methionine heme ligand resonance of the reduced form of the apoenzyme. On the other hand, resonance Raman spectra of the heme in the different enzyme forms were nearly similar. These results suggest that a major effect of PQQ binding to apo-QH-EDH is a rotation of the methionine ligand of heme c. Since no intermediate 1H-NMR spectra were observed upon titration of apoenzyme with PQQ, apparently no exchange occurs of PQQ between (oxidized) holo- and apoenzyme at the NMR time scale and at that of the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

The stearoyl-ACP delta 9 desaturase from plants is a new example of a growing number of proteins that contain oxo- or hydroxo-bridged diiron clusters. On the basis of differences in primary sequence motifs providing the cluster ligands and upon structural differences elucidated by X-ray crystallography, we now propose that the presently known, soluble diiron-oxo proteins can be grouped into two classes, I and II. Class I contains hemerythrin, myohemerythrin, and, possibly, purple acid phosphatase. Class II contains ribonucleotide reductases, bacterial hydrocarbon hydroxylases (methane monooxygenase, toluene-4-monooxygenase, and phenol hydroxylase), rubrerythrin, and stearoyl-ACP desaturases. Through the use of resonance Raman spectroscopy, we have detected symmetric (vs = 519 cm-1) and asymmetric (vas = 747 cm-1) vibrational modes in the castor stearoyl-ACP delta 9 desaturase, which are typical of oxo-bridged diiron clusters. These frequencies shift by -18 and -34 cm-1, respectively, in H218O, proving that the bridging ligand is readily exchangeable with solvent (t1/2 = 7 min). Calculation of an approximately 123 degrees Fe-O-Fe angle from the position of vs and vas and from the 18O-dependent shift in these frequencies suggests that the diiron-oxo cluster in the desaturase is triply bridged in the diferric state. In the diferrous state, the two iron sites of the cluster are structurally inequivalent, as shown by differential temperature dependence of the Mossbauer quadrupole splittings. For the class II diiron-oxo proteins, primary sequence alignments reveal conserved amino acid residues which act as iron cluster ligands, participate in a hydrogen-bonding network, and are potentially involved in O2 binding and activation. Based on this conservation, a structural model for the stearoyl-ACP delta 9 desaturase active site is proposed that has strong similarity to both ribonucleotide reductase and methane monooxygenase. However, after single turnover of the diferous state with 18O2, 18O is not detected in the oxo bridge of the castor desaturase. This is in contrast to the outcome observed for ribonucleotide reductase, suggesting the desaturase and ribonucleotide reductase differ in certain aspects of their respective O2-activation reactions.

Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 1H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (E(m) = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the alpha carbon of the heme 4-vinyl group. Form BI has a covalently bound heme group coupled through a thioether linkage involving the beta carbon of the heme 4-vinyl group. Form BII is similar to BI except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (E(m) = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed.

Publisher Summary The focus of this chapter is pragmatic. It provides tested laboratory procedures that discuss the way resonance Raman spectra of metalloproteins are obtained and verified, the way active-site structural and/or mechanistic information is probed by isotopic labeling, and the way spectral data interpretation is initiated. The specific-methods approach given here discusses sample preparation and temperature control, choice of scattering geometries, data collection, data quality, and the initiation of data analysis. It describes common precautions and pitfalls, as well as criteria for judging sample integrity. It discusses the way one confirms the resonance Raman phenomenon and also discusses the utility of excitation profiles. The general theory of Raman spectroscopy and resonance Raman spectroscopy in particular, including a description of Raman instrumentation, laser sources, and general applications of the technique, is presented in this chapter. Raman scattering is a relatively weak phenomenon and therefore requires high sample concentrations, but samples may be studied in all phases of matter.

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.

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.

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.

The active form of protein B2, the small subunit of ribonucleotide reductase, contains two dinuclear Fe(III) centers and a tyrosyl radical. The inactive metB2 form also contains the same diferric complexes but lacks the tyrosyl radical. We now demonstrate that incubation of metB2 with hydrogen peroxide generates the tyrosyl radical. The reaction is optimal at 5.5 mM hydrogen peroxide, with a maximum of 25–30% tyrosyl radical being formed after approximately 1.5 hr of incubation. The activation reaction is counteracted by a hydrogen peroxide-dependent reduction of the tyrosyl radical. It is likely that the generation of the radical proceeds via a ferryl intermediate, as in the proposed mechanisms for cytochrome P-450 and the peroxidases.

Site-directed mutagenesis offers a powerful probe of heme proteins. Recent attention has focused on mutants whose proximal iron ligand is replaced by smaller, noncoordinating amino acids. These mutants form artificial cavities where the native proximal ligand resided and which are capable of binding exogenous ligands such as imidazoles. Such reconstituted systems permit detailed studies of the electronic and stereochemical influences of the proximal ligand on catalysis and activity.1-6 The paradigm of these mutants is H175G cytochrome c peroxidase (CCP). Its X-ray crystal structure revealed that the iron is close to two water molecules.1 In heme oxygenase, the H25A mutant has been proposed to contain a single water ligand on the heme iron.4 The iron in the H170A mutant of horseradish peroxidase is probably bound by a water molecule at pH 4 and an additional sixth ligand (possibly a distal histidine) at pH 5-7.6 The crystal structure of the imidazole adduct of H93G myoglobin has been reported,2 but structural and spectroscopic data for the H93G mutant in the absence of imidazole are not yet available. Interestingly, UV-vis spectra of wild-type metmyoglobin and ferric Coprinus cinereus peroxidase in acidic buffer7,8 bear resemblance to those of the proximal ligand-deficient heme proteins; the native proximal ligation may well be disrupted under such conditions. Among these proximal-ligand mutants, H175G CCP is unique in that its crystal structure indicates two water molecules axial to the heme. However, it is not certain whether one or both of these waters is strongly coordinated, what the water protonation states are, and whether the axial coordination of two water molecules can be maintained in solution. In this study, we have examined the crystal and solution structures of the H175G mutant by UVvis, resonance Raman (RR), and EPR spectroscopy. Ferric H175G CCP9 undergoes a transition from a “red form” near pH 6 to a “green form” near pH 7 (Figure 1). The absorbance at 408 nm (λmax of the red form) changes with pH to give an apparent pKa of ∼6.5 (inset, Figure 1). A fit to these data indicates that the transition involves a cooperative twoproton process. One proton is proposed to be from the ironbound water molecule, but the source of the second proton is unknown. The UV-vis and RR spectra of H175G at pH 10.0 are almost identical to those at pH 7.2 (not shown). FeIII(TMP)s [TMP ) 5,10,15,20-tetrakis(1-methylpyridinium)porphines] exhibit similar ligand ionization in aqueous solution where the five-coordinate iron is ligated by a water at pH 11.10,11 Resonance Raman spectra of heme proteins are dominated by porphyrin skeletal vibrations, i.e., totally symmetric ν2, ν3, and ν4 with Soret excitation, nonsymmetric modes ν10, ν11, and ν19 with Q-band excitation, and ν2 and ν10 with near-UV (∼350 nm) excitation.12-15 The frequencies of these modes are determined by the iron coordination and spin states. The RR spectra of H175G16 are shown in Figure 2. The well-defined ν3 and ν10 frequencies at 1494 and 1630 cm-1, respectively, of the red solution (pH 5.9) are clearly characteristic of a fivecoordinate high-spin (5cHS) ferric heme.15,17-20 The RR spectra of the green solution (pH 7.2) also indicate a dominant 5cHS

The resonance Raman spectrum of protein B2 of ribonucleotide reductase from Escherichia coli shows several features related to its oxo-bridged binuclear iron center. A peak at 492 cm/sup -1/ is assigned to the symmetric stretch of the Fe-O-Fe moiety on the basis of its 13-cm/sup -1/ shift to lower energy upon /sup 18/O substitution. The /sup 18/O species shows an additional peak at 731 cm/sup -1/, which is a good candidate for the asymmetric stretch of the Fe-O-Fe moiety. Its exact location in the /sup 16/O species is obscured by the presence of a protein tryptophan vibration at 758 cm/sup -1/. A third resonance-enhanced peak at 598 cm/sup -1/ is identified as an Fe-OH vibration on the basis of its 24-cm/sup -1/ shift to lower energy in H/sub 2//sup 18/O, its 2-cm/sup -1/ shift to lower energy in D/sub 2/O, 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/sup -1/ shift to higher energy of nu/sub s/ (Fe-O-Fe), an Fe-O-Fe angle of 138/sup 0/ can be calculated. This small angle suggests that the iron center consists of a tribridged core asmore » 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.« less

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.

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.

Resonance Raman spectroscopy has been used to study milk xanthine oxidase, an enzyme containing molybdenum, binuclear iron-sulfur clusters, and FAD as cofactors. The contribution of FAD dominates the resonance Raman spectrum at frequencies above 500 cm-1. As expected, no bands assignable to FAD are observed in deflavo xanthine oxidase. The resonance Raman spectrum below 500 cm-1 reveals the contribution of the Fe2S2(Cys)4 groups with frequencies similar to those of adrenodoxin and putidaredoxin. Resonance enhancement profiles of the Fe2S2(Cys)4 clusters indicate intensity variations among the Fe2S2(Cys)4 peaks that are attributed to different excitation wavelength maxima of their bridging and terminal iron-sulfur vibrations. No evidence for Mo-ligand vibrations could be obtained by using excitation wavelengths between 363.8 and 514.5 nm.

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.

The Raman spectrum of protocatechuate 3,4-dioxygenase [EC 1.13.11.3] shows four principal resonance-enhanced peaks at 1602, 1503, 1263 and 1171 cm −1 with 514.5 nm laser excitation. These frequencies are associated with ringmode vibrations of one or more tyrosinate residues coordinated with the Fe(III) at the active site. These data provide the first direct evidence for the identity of a permanent iron ligand in this enzyme. The great similarity in the resonance Raman spectrum of protocatechuate 3,4-dioxygenase with those of iron-transferrins suggests the existence of a class of proteins characterized by Fe(III)-tyrosinate coordination.

The i.r. and Raman spectra of the N-coordinated copper(II)— and copper(III)—bis(biuretato) and —bis(oxamidato) complexes have been recorded, and normal coordinate analyses have yielded a Urey—Bradley force field for the Cu(III) complexes. The biuret system undergoes little change on oxidation, characterized by ring tightening and a general increase in mode frequencies. On oxidation, the oxamide system undergoes more drastic structural changes, most notably a strengthening of the CC bond in the oxamide rings, as shown by a 19% increase in K(CC), and an increase in electron density in the OCCO framework.

Raman spectra are reported for liquid and solid chromyl fluoride. Nine Raman lines, of which four are polarized, are observed for CrO2F2(l) and assigned to a monomeric structure possessing C2v symmetry. Some reassignments for previously reported gas phase infrared bands are suggested based on the new Raman data. The symmetric Cr–F stretching vibration at 708 cm−1 in CrO2F2(l) disappears in the spectrum of the solid phase material, which has a new Raman line at 540 cm−1 characteristic of extensive fluorine bridge bonding.

Resonance Raman, optical absorption, circular dichroic, and fluorescence emission spectroscopy of hemerythrins from four species of sipunculids (Phascolopsis gouldii, Phascolosoma agassizii, Themiste dyscritium, and Themiste pyroides) reveals no major differences in their active site or tertiary structures. This precludes any change in iron ligands or coodination geometry and makes it unlikely that the active-site structures of P. gouldii and T. dyscritum hemerythrins could be as disparate as indicated by present crystallographic interpretations (Stenkamp, R. E., Sieker, L. C., and Jensen, L. H. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 349; Klotz, I. M., Klippenstein, G. L., and Hendrickson, W. A. (1976), Science 192, 335). Resonance Raman enhancement profiles of the stretching modes involving coordinated dioxygen maximize with excitation at approximately 525 nm, and correspond to the circular dichroic (CD) transition at approximately 520 nm. For coordinated azide modes in metazidohemerythrins these profiles maximize with excitation at approximately 505 nm corresponding to the 500-nm CD transition. Hemerythrins also possess another resonance Raman peak at approximately 510 cm-1 which show maximum intensity enhancement at approximately 530 nm and this vibration is most likely associated with a permanent iron ligand.

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.

Resonance Raman spectra of the complex of Fe/sup III/-ethylenediaminetetraacetate with hydrogen peroxide have been obtained in the region of the peroxidic O-O stretching vibration that occurs at 815 cm/sup /minus/1/ in frozen solution. When the complex was prepared from nearly neat, mixed-isotope hydrogen peroxide, H/sub 2//sup 16/O/sup 18/O, a single O-O vibration was observed at 794 cm/sup /minus/1/. This observation establishes that the two oxygen atoms of the bound peroxide are equivalent and that the peroxide is ligated in an /eta//sup 2/ side-on configuration in this mononuclear complex. The absence of any shift in /nu/(O-O) when the complex was prepared in D/sub 2/O solvent provides further support for the side-on Fe(O-O) geometry and rules out the alternative /eta//sup 1/ end-on Fe-OOH configuration. In contrast, /nu/(O-O) of free hydrogen peroxide in the frozen state occurs at 877 cm/sup /minus/1/ in H/sub 2/O and 879 cm/sup /minus/1/ in D/sub 2/O. The 2-cm/sup /minus/1/ upshift in D/sub 2/O is similar to that observed previously with oxyhemerythrin and appears to be characteristic of a hydrogen-bonded or protonated peroxide species. 37 references, 4 figures, 1 table.

The Raman and i.r. spectra of the dioxomolybdenum(VI) complexes, MoO2[NH2CH2C(CH3)2S]2, 1, MoO2[CH3NHCH2C(CH3)2S]2, 2, MoO2[(CH3)2NCH2C(CH3)2S]2, 3, and MoO2[(SCH2CH2)2NCH2CH2N(CH3)2], 4, have been investigated in the region of the MoO2 stretching frequencies. 18O substitutions for the oxygen atoms in these complexes gave rise to complicated patterns in their vibrational spectra. A normal coordinate analysis has been applied to interpret the vibrational modes of complex 1 in terms of coupling the symmetric and antisymmetric OMoO stretches. Where the O atoms are inequivalent, the frequency differences between pairs of coupled modes varied with the site(s) of the isotopic substitution(s). The particular pattern of frequency differences was then used to aid in the spectral assignments of the remaining complexes.

Etude par spectroscopie de resonance Raman des complexes, Fe III (photoporphyrine IX ester dimethyl) et Fe III (deuterochlorine IX ester dimethyl)

Reactions of chromium pentafluoride, CrF5, with some Lewis acids and bases have been studied and the reaction products have been characterized by infrared and Raman spectroscopy. CrF5reacts with the bases CsF and NO2F to give CsCrF6 and NO2CrF6, respectively. The Lewis acid, SbF5, reacts with CrF5 to give the adduct CrF5·2SbF5, which should be formulated as CrF4Sb2F11 on the basis of spectroscopic evidence. This compound is a strong oxidizing agent, reacting with O2, Xe and C6F6 to form, respectively, O2(CrF4Sb2F11), Xe(CrF4Sb2F11)2 and C6F6(CrF4Sb2F11). It was observed that O2(CrF4Sb2F11) reacts with C6F6 and IF5 to form C6F6(CrF4Sb2F11) and IF4(CrF4Sb2F11), respectively.

Resonance Raman spectroscopy was used to investigate the Ni-tetrapyrrole, cofactor F430, of methanogenic bacteria. Excitation within the 430-nm absorption band produced resonance-enhanced vibrational modes and a spectral pattern that contrasts with resonance Raman data of other metal-tetrapyrrole complexes (heme-, metalloporphyrin- and vitamin B-12-derivatives). With 406.7-nm excitation, the most intense spectral features were observed at 1530, 1628 and 1562 cm−1. Although many weaker spectral bands were recorded below 1500 cm−1, the relative simplicity of the F430 resonance Raman spectrum is in accord with the lower degree of unsaturation in the nickel complex. Excitation at longer wavelengths brought several low-frequency modes in the 200–500 cm−1 region into resonance. This observation indicates that the 430-nm band is composed of multiple electronic transitions. The low-frequency spectrum, which should contain metal-ligand vibrations, may be particularly useful in studying the interactions of F430 with coenzyme M and methylreductase.

Cristallisation dans le groupe P1. Affinement jusqu'a R=0,0895. Le cation dimere montre une longueur de liaison Fe−O normale, 1,785 A et un angle Fe−O−Fe de 155,1°. Presentation detaillee des spectres IR et Raman pour l'etude de la vibration Fe−O−Fe

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 [Sjoberg, 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.

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 ∼ 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.

The green heme protein sulfmyoglobin (SMb) has been suggested to contain a sulfur-modified iron chlorin prosthetic group. To evaluate this hypothesis, we have obtained high-frequency (greater than 1000 cm-1) resonance Raman spectra of both oxidized and reduced SMb with 457.9-, 488.0-, 514.5-, 568.2-, and 647.1-nm excitation. The SMb spectra are compared to those of native met- and deoxymyoglobin (Mb). Vibrational frequencies for SMb are generally similar to those of Mb, suggesting a high-spin state for both the Fe(III) and Fe(II) SMb species, as is typical of native Mb. However, major differences between SMb and Mb occur both for patterns of relative spectral intensities and for depolarization ratios. In particular, all B1g-depolarized porphyrin modes in the Mb spectra have become polarized, totally symmetric vibrational modes in the SMb spectra. These contrasts reflect a dramatic lowering of the effective symmetry for the SMb prosthetic group. Several new bands are observed in SMb spectra that are not present in spectra of either native Mb or iron protoporphyrin IX complexes. The observation of additional polarized bands flanking the oxidation state marker, V4, is of particular interest. In a parallel study, we compared the resonance Raman spectral properties of iron protoporphyrin IX-derived chlorins and metallo-octaethylchlorins with those of the analogous porphyrins: the chlorin spectra exhibited altered intensity patterns, an increased number of totally symmetric (polarized) vibrational bands, and several new vibrational bands, including one or two in the region of the oxidation state marker, V4. Thus, the resonance Raman spectral characteristics of SMb and metallo-chlorins are complementary and strongly support a chlorin prosthetic group for SMb. Furthermore, they establish testable criteria for investigating the prosthetic group structures of other green heme proteins by resonance Raman spectroscopy.

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.

The Raman spectrum of the B2 subunit of Escherichia coli ribonucleotide reductase shows a peak at 496 cm-1 that appears to be in resonance with the 370-nm electronic transition of the binuclear iron center in both the native and radical-free forms of the protein. Exposure of the protein to H218O causes the peak to shift to 481 cm-1, indicating that the vibrational mode is due to an Fe-O moiety in which the oxygen can exchange with solvent. The rate of oxygen exchange (kobsd = 8.3 x 10-4 s-1) is consistent with a mu-oxo-bridged structure. Protonation of the oxygen is unlikely since the Fe-O vibration fails to shift to lower frequency in D2O. Instead, there is a gradual increase in the vibrational frequency with time to a maximum value of 502 cm-1 after 3 h in 70% with time to a maximum value of 602 cm-1 after 3 h in 70% D2O. Apparently, the deuteration of successive protein functional groups causes a slight alteration in the structure of the binuclear iron center. The resonance Raman characteristics of the Fe-O-Fe group in protein B2 are similar to those previously reported for the mu-oxo-bridged binuclear iron center in hemerythrin. A further similarity between the two proteins is the high degree of alpha-helical content. Circular dichroism measurements place this value at approximately 60% for the B2 subunit of ribonucleotide reductase.

Normal coordinate analysis that utilizes a general valence force field and the Wilson FG matrix method has been applied to several structural models representing the active site of the blue copper protein, azurin. The models included tetrahedral and square planar CuN2SS', trigonal CuN2S, and trigonal bipyramidal CuN2SS'O structures in which the Ns are imidazole nitrogens of histidines, S is the thiolate sulfur of cysteine, S' is the thioether sulfur of methionine, and O is a peptide carbonyl oxygen. For constant Cu--ligand bond lengths and initial force constants, the force field was refined against the most intense of the observed frequencies (424, 404, 369, and 261 cm-1) in the resonance Raman spectrum of Pseudomonas aeruginosa azurin. The most satisfactory fit between observed and calculated frequencies occurs for tetrahedral and trigonal structures. The calculations provide detailed assignments for the resonance Raman spectrum of azurin and reveal considerable mixing of Cu--S(Cys) and Cu--N(His) vibrational modes. The trigonal model is favored because it is shown that the approximately equal to 260-cm-1 vibration is an invariant feature in the resonance Raman spectra of blue copper proteins, even those lacking a methionine in the vicinity of the copper atom. The present analysis ascribes the high frequencies of the Cu--ligand stretching modes and the resonance enhancement to the coupled nature of their vibrations and the Franck-Condon overlaps with predominant (Cys)S leads to Cu(II) charge transfer bands in the visible region.