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Cystathionine beta-synthase is a key heme and pyridoxal phosphate-dependent enzyme involved in homocysteine metabolism in humans. The role of the recently discovered heme in this protein remains an important open question. The axial ligands to the heme in both the ferrous and ferric states have been assigned as cysteine and histidine residues, respectively. In this study, we have examined the effect of ligation and spin state changes in the heme on the activity of the enzyme. Treatment of the ferric enzyme with HgCl2 results in the conversion of six-coordinate low-spin heme to five-coordinate high-spin heme and is paralleled by a loss of activity. In contrast, treatment of the ferrous enzyme with HgCl2 results in replacement of the cysteine ligand by an unidentified sixth ligand and retention of the six-coordinate state, and is also accompanied by loss of enzyme activity. Treatment of the five-coordinate HgCl2-treated enzyme with thiols, such as homocysteine, results in reversion to a six-coordinate state. Resonance Raman spectroscopy with 34S-labeled enzyme reveals the return of the endogenous thiol ligand under these conditions and rules out direct coordination by the thiolate of homocysteine to the heme.

The crystal structure of heme oxygenase-1 suggests that Asp-140 may participate in a hydrogen bonding network involving ligands coordinated to the heme iron atom. To examine this possibility, Asp-140 was mutated to an alanine, phenylalanine, histidine, leucine, or asparagine, and the properties of the purified proteins were investigated. UV-visible and resonance Raman spectroscopy indicate that the distal water ligand is lost from the iron in all the mutants except, to some extent, the D140N mutant. In the D140H mutant, the distal water ligand is replaced by the new His-140 as the sixth iron ligand, giving a bis-histidine complex. The D140A, D140H, and D140N mutants retain a trace (

CYP119 from Sulfolobus solfataricus, the first thermophilic cytochrome P450, is stable at up to 85 degrees C. UV-visible and resonance Raman show the enzyme is in the low spin state and only modestly shifts to the high spin state at higher temperatures. Styrene only causes a small spin state shift, but T(1) NMR studies confirm that styrene is bound in the active site. CYP119 catalyzes the H(2)O(2)-dependent epoxidation of styrene, cis-beta-methylstyrene, and cis-stilbene with retention of stereochemistry. This catalytic activity is stable to preincubation at 80 degrees C for 90 min. Site-specific mutagenesis shows that Thr-213 is catalytically important and Thr-214 helps to control the iron spin state. Topological analysis by reaction with aryldiazenes shows that Thr-213 lies above pyrrole rings A and B and is close to the iron atom, whereas Thr-214 is some distance away. CYP119 is very slowly reduced by putidaredoxin and putidaredoxin reductase, but these proteins support catalytic turnover of the Thr-214 mutants. Protein melting curves indicate that the thermal stability of CYP119 does not depend on the iron spin state or the active site architecture defined by the threonine residues. Independence of thermal stability from active site structural factors should facilitate the engineering of novel thermostable catalysts.

Cellobiose dehydrogenase (CDH), an extracellular hemoflavoenzyme produced by cellulose-degrading cultures ofPhanerochaete chrysosporium,oxidizes cellobiose to cellobionolactone. The enzyme contains one 6-coordinate, low-spinb-type heme and one FAD cofactor per monomeric protein. In this work, resonance Raman (RR) spectra are reported for the oxidized, reduced, and deflavo forms of CDH as well as the individual flavin and heme domains of the enzyme obtained by peptide proteolysis. The RR spectra of the flavin and heme groups of CDH were assigned by comparison to the spectra of other hemoflavoenzymes and model compounds. Proteolytic cleavage of the CDH domains had only a minimal spectroscopic effect on the vibrational modes of the heme and FAD cofactors. Excitation of the oxidized CDH holoenzyme at 413 or 442 nm resulted in photoreduction of the heme. However, the same excitation wavelength used on the deflavo form of the enzyme or on the heme domain alone did not cause photoreduction, indicating that photoinitiated electron transfer requires the FAD cofactor. These observations suggest an enzymatic mechanism whereby reducing equivalents obtained from the oxidation of cellobiose are transferred from the FAD to the heme. A similar mechanism has been proposed for flavocytochrome b2ofSaccharomyces cerevisiaewhich oxidizes lactate to pyruvate (A. Desboiset al.,1989,Biochemistry28, 8011–8022).

Conversion of heme to verdoheme by heme oxygenase-1 (HO-1) is thought to involve alpha-meso-hydroxylation and elimination of the meso-carbon as CO, a reaction supported by both H2O2 and NADPH-cytochrome P450 reductase/O2. Anaerobic reaction of the heme-HO-1 complex with 1 eq of H2O2 produces an enzyme-bound intermediate identified by spectroscopic methods as alpha-meso-hydroxyheme. This is the first direct evidence for HO-1-catalyzed formation of alpha-meso-hydroxyheme. alpha-meso-Hydroxyheme exists as a mixture of Fe(III) phenolate, Fe(III) keto anion, and Fe(II) keto pi neutral radical resonance structures. EPR shows that complexation with CO enhances the Fe(II) pi neutral radical component. Reaction of the alpha-meso-hydroxyheme-HO-1 complex with O2 generates Fe(III) verdoheme, which can be reduced in the presence of CO to the Fe(II) verdoheme-CO complex. Thus, conversion of alpha-meso-hydroxyheme to Fe(III) verdoheme, in contrast to a previous report (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624), does not require a reducing equivalent. An electron is only required to reduce ferric to ferrous verdoheme in the first step of its conversion to biliverdin.

Vibrational spectra of tetracoordinate Cu(II)-dimethyloctaethylisobacteriochlorins (diMeOEiBC) were studied. The complexes include: 2,4-diMeOEiBC (1) and its 15 N and d 6 isomers ( meso-d 2 ′4′- d 2 ), 2,3-diMeOEiBC (2), 1,3-diMeOEiBC (3), and 2′,4′-diOH-2,4-diMeOEiBC (4). The generalized resonance Raman (RR) and Fourier transform infrared (FTIR) spectral properties of the diMeOEiBCs are similar to those of low-symmetry (~ C 2 ) metallochlorins, and relative to porphyrins, display an increase in the number of vibrational bands, considerable congruence between RR and FTIR frequencies, an increase in the number of totally symmetric RR bands, a cluster of bands in the ~1340–1420 cm −1 region, and an intense FTIR band >1600cm −1 .In the ~1340–1420cm −1 region, iBC complexes display their dominant band ≥1400cm −1 , increasing in intensity with excitation towards the red. This feature, which has its maximum at the position of the Q y ,(0,0) band, as shown by an excitation profile for the 2,4-diMeOEiBC macrocycle, is essentially insensitive to 15 N 4 or d 6 ( meso - d 4 -2′, 4′- d 2 ) substitution and is likely to be derived from the porphyrin υ 29 mode. The RR data reflect differences in identity/pattern of the pyrroline (saturated) ring substituents, e.g. 2,4-diMeOEiBC has bands at 1650 and 1620 cm −1 ; vs 1648 and 1610 cm −1 for 2′,4′-diOH-2,4-diMeOEiBC; vs 1649, 1640 and 1617 cm −1 for 2,3-diMeOEiBC; and vs 1645 and 1610 cm −1 for 1,4-diMeOEiBC. Thus, just as for metallochlorins [Andersson et al., Inorg. Chem. 29 , 2278 (1990)], localized structural differences in non-conjugated pyrroline substituents of metallo-isobacteriochlorins can affect the resonance Raman spectral properties dramatically.

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.

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.

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.

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.

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.

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.

Schizokinen, a citrate-containing dihydroxamate, is a siderophore produced by Bacillus megaterium and Anabaena sp. The involvement of the citrate α-hydroxycarboxylate moiety in iron chelation was investigated by comparing the iron binding behavior of schizokinen with that of acetylschizokinen, a derivative in which the citrate hydroxyl group was modified by acetylation. Ferric schizokinen was found to exhibit an absorption spectrum (λmax = 460 nm) characteristic of a dihydroxamate below pH 2.5, with an isosbestic shift to a citrate dihydroxamate spectrum (λmax = 395 nm) above pH 4. Ferric acetylschizokinen also had a dihydroxamate absorption spectrum (λmax = 465 nm) at low pH. However, its spectral shift (λmax = 420 nm) and intensity above pH 4 were more typical of a ferric trihydroxamate. The molecular weight and electrophoretic mobility of ferric acetylschizokinen are consistent with a dimeric Fe2 (acetylschizokinen)3 structure, whereas ferric schizokinen appears to exist as a monomeric 1:1 complex Despite the differences in molecular weight and α-hydroxycarboxylate coordination, both complexes are effective in promoting iron uptake in Anabaena.

Binuclear iron centers are known to be present in the respiratory protein, hemerythrin [1], and in the enzyme, ribonucleotide reductase [2]. These centers are characterized by strong antiferromagnetic coupling (-J ≅ 100 cm −1 ) of the two ferric ions, and by one or more intense absorption bands between 320 and 380 nm (ϵ ≅ 4000 M −1 cm − per Fe atom). These properties have long been ascribed to the presence of a μ-oxo bridge between the iron atoms. Verification of such a bridge was obtained in the 2.2-A resolution crystal structure of azidomethemerythrin [3]. Resonance Raman spectroscopy provides an additional valuable technique for the detection and characterization of binuclear iron centers. The FeOFe symmetric stretch, ν s (FeOFe), is Raman active and the intensity of this vibration may be enhanced by excitation within the FeOFe charge transfer band in the near ultraviolet. As a model system, we have investigated the resonance Raman spectra of binuclear 1,10-phenanthroline (phen) complexes of iron(III) [4]. The complex Fe 2 O(phen) 4 (NO 3 ) 4 · 7H 2 O has a Raman peak at 395 cm −1 which can be assigned to ν s (FeOFe) on the basis of its frequency being appropriate to an FeOFe angle of 154°, its absence from the spectrum of mono-nuclear complex Fe(phen) 3 (ClO 4 ) 3 · 3H 2 O, and its intensity being dependent upon excitation wavelength. The perchlorate and chloride salts of [Fe 2 O(phen) 4 ] 4+ have similar resonance-enhanced modes close to 400 cm −1 , and in all three cases the enhancement is maximized using 363.8 nm excitation. Confirmatory evidence for an FeOFe vibration can be obtained from oxygen isotope exchange with H 18 2 O solvent. For example, in the μ-oxo bridged dimer, [Fe 2 O(Cl) 6 ] 2− , ν s (FeOFe) at 458 cm −1 shifts to 400 cm −1 in H 2 18 O [5]. Similarly, the resonance-enhanced band at 507 cm −1 in the Raman spectrum of azidomethemerythrin shifts to 490 cm −1 for a sample which has been formed from oxyhemerythrin H 18 2 O [6]. We have now found evidence for the corresponding FeOFe vibration at 489 cm −1 in the resonance Raman spectrum of oxyhemerythrin, through the use of near-ultraviolet excitation [7]. Furthermore, this band shows an isotope dependence on solvent (H 18 2 O), and appears to be in resonance with the 360 nm electronic transition of oxyhemerythrin. The resonance enhancement of the ν s (FeOFe) peak intensity with ultraviolet excitation has also been observed for azidomethemerythrin [7] and for ribonucleotide reductase [2]. In contrast to hemerythrin, however, where the oxo group only exchanges during exogenous ligand replacement, the oxo group in ribonucleotide reductase undergoes facile exchange with solvent (k obs = 8.3 × 10 −4 s −1 , indicating it is located in a more accessible site.

‘Blue’ copper proteins possess intense visible absorption bands at ≅600 nm due to (Cys)S − → Cu(II) charge transfer. The type 1 copper in these proteins also exhibits anomalously low hyperfine splitting in the EPR spectra and high redox potentials. Crystallographic and EXAFS investigations on two simple blue copper proteins, plastocyanin and azurin, have provided fairly detailed pictures on their active site structures. The resonance Raman (RR) spectra of blue copper proteins are remarkably similar to one another; all have three or more strong bands in the 350–450 cm −1 range; all exhibit intensity enhancement by resonance with the ≅600 nm LMCT band; all spectra have two nearly constant frequency components at ≅750 and ≅250 cm −1 which appear to be vibrational modes of the coordinated cysteinate ligand; and, finally, many weaker spectral features are resolvable in high quality spectra (Fig. 1). The complexity of the RR spectra of the type 1 copper sites suggests the vibrational modes of the chromophore in addition to CuS(Cys) stretching must also contribute. Interpretation is complicated by the fact that the principal peaks in the RR spectra are at unprecedently high frequencies for copper complexes involving sulfur and nitrogen coordination. We recently undertook a normal coordinate analysis of the CuN 2 S(S′) chromophore structure of Pseudomonas aeruginosa azurin. We found that the anomalously high frequencies can be explained by the shortness of the CuS (cysteinate) bond and the t001 . Raman Frequencies (cm −1 ) for CuLADH at 77 K. Protein ν a ν b ν c ν d CuLADH 415 350 254 199 + NADH 420 359 263 202 + pyrazole 413 368 246 196 strongly coupled nature of the vibrations. Good agreement with observed azurin frequencies was obtained for a trigonal pyramidal model with Cu(II) coordinated to two histidines and one cysteinate [1]. The structure of the zinc enzyme, liver alcohol dehydrogenase (LADH), is well documented. Substitution of the catalytic zinc by Cu(II) yields a protein that has type 1 copper properties. We have investigated the RR spectrum of this system and find it to be an excellent model for the spectra of blue copper proteins [2]. A spectrum of CuLADH is shown in Fig. 1. The main spectral features are also at high frequencies and correlate well with those observed for azurin. The catalytic Zn(II) ion in LADH is coordinated by two cysteinates, one histidine, and one water molecule in a tetrahedral environment. We attribute the sharp Raman peak at 415 cm −1 in CuLADH to a predominantly Cuhistidine vibration and the broader band at ≅ 350 cm −1 to contributions from the two Cu-cysteinates. The high frequencies of the chromophore vibrations lead to the prediction that the CuS bonds distances in CuLADH are also close to 21.1 A, as in azurin and plastocyanin. Addition of the coenzyme, NADH, or pyrazole, a competitive inhibitor of alcohol oxidation in the native enzyme, to the copper proteins causes only minor changes in the RR spectrum of the Cu(II) chromophore (Table I). Thus, despite the large shift of the absorbance maximum from 580 to 495 nm upon pyrazole binding, the copper site appears to maintain its near tetrahedral geometry. Since normal coordinate analyses indicate that vibrational frequencies are sensitive to geometric variations in this type of system [1], any structural changes responsible for the altered λ max and hyperfine splitting in the Cu-LADH/pyrazole complex must be fairly subtle ones.