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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 (

Human cystathionine beta-synthase (CBS) is an essential enzyme for the removal of the toxic metabolite homocysteine. Heme and pyridoxal phosphate (PLP) cofactors are necessary to catalyze the condensation of homocysteine and serine to generate cystathionine. While the role for the PLP cofactor is thought to be similar to that in other PLP-dependent enzymes that catalyze beta-replacement reactions, the exact role for the heme remains unclear. In this study, we have characterized the heme cofactor of CBS in both the ferric and ferrous states using resonance Raman spectroscopy. Positive identification of a cysteine ligand was achieved by global (34)S isotopic substitution which allowed us to assign the nu(Fe-S) for the six-coordinate low-spin ferric heme at 312 cm(-1). In addition, the CO adduct of ferrous CBS has vibrational frequencies characteristic of a histidine-heme-CO complex in a hydrophobic environment, and indicates that the Fe-S(Cys) bond is labile. We have also found that addition of HgCl(2) to the ferric heme causes conversion of the low-spin heme to a five-coordinate high-spin heme with loss of the cysteine ligand. The present spectroscopic studies do not support a reaction mechanism in which homocysteine binds directly to the heme via displacement of the Cys ligand in the binary enzyme complex, as had been previously proposed.

Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from approximately 123 degrees in the oxo-bridged diferric centers of the as-isolated enzyme to approximately 120 degrees in oxo-bridged diferric centers of the complex. Analysis of the shifts in nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta9D was used to investigate whether an O-atom from O(2) would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of approximately 70% yield of 18:1-ACP product in the presence of (18)O(2), no incorporation of an (18)O atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O(2) into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta9D catalysis are discussed.

Manganese peroxidase (MnP) from Phanerochaete chrysosporium undergoes a pH-dependent conformational change evidenced by changes in the electronic absorption spectrum. This high- to low-spin alkaline transition occurs at approximately 2 pH units lower in an F190I mutant MnP when compared to the wild-type enzyme. Herein, we provide evidence that these spectral changes are attributable to the formation of a bis(histidyl) heme iron complex in both proteins at high pH. The resonance Raman (RR) spectra of both ferric proteins at high pH are similar, indicating similar heme environments in both proteins, and resemble that of ferric cytochrome b(558), a protein that contains a bis-His iron complex. Upon reduction with dithionite at high pH, the visible spectra of both the wild-type and F190I MnP exhibit absorption maxima at 429, 529, and 558 nm, resembling the absorption spectrum of ferrous cytochrome b(558). RR spectra of the reduced wild-type and F190I mutant proteins at high pH are also similar to the RR spectrum of ferrous cytochrome b(558), further suggesting that the alkaline low-spin species is a bis(histidyl) heme derivative. No shift in the low-frequency RR bands was observed in 75% (18)O-labeled water, indicating that the low-spin species is most likely not a hydroxo-heme derivative. Electronic and RR spectra also indicate that addition of Ca(2+) to either the ferric or ferrous enzymes at high pH completely restores the high-spin pentacoordinate species. Other divalent metals, such as Mn(2+), Mg(2+), Zn(2+), or Cd(2+), do not restore the enzyme under the conditions studied.

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.

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies. The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.

Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mössbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mössbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.

cDNAs coding for bovine endothelial nitric oxide synthase (eNOS) with N-terminal deletions of 52, 91, and 105 amino acids were constructed, and the proteins were expressed in Escherichia coli and purified by affinity chromatography. All three truncated proteins bind heme and exhibit the ferrous-CO absorption maximum at 444 nm characteristic of thiolate heme ligation. Deletion of the first 52 amino acids yields a fully active dimeric protein with the same spectroscopic properties as the wild-type. The myristoylation, palmitoylation, and polyproline domains of the enzyme located in the deleted region are therefore not required for full catalytic activity. The delta91 and delta105 proteins, which exhibit altered dimerization equilibria, retain 20 and 12%, respectively, of the maximal activity. Resonance Raman and UV-vis spectroscopy indicate that, in the absence of tetrahydrobiopterin (H4B) and l-Arg, the wild-type and delta52 proteins are predominantly five coordinate high spin, whereas the delta91 and delta105 proteins are six coordinate low spin. The delta91 and delta105 mutants bind H4B, as indicated by a concomitant decrease in the low-spin component of the UV-vis spectrum, but the binding of l-Arg is extremely slow ( approximately 15 min). Dithiothreitol readily coordinates as the sixth iron ligand in the delta91 and delta105 mutants but not in the delta52 or wild-type proteins. The dithiothreitol can be completely displaced by l-Arg but not by H4B. Resonance Raman comparison of wild-type eNOS and nNOS confirms that, in the absence of H4B and l-Arg, eNOS is primarily high spin whereas nNOS is predominantly six coordinate, low spin. The results indicate that Cys-101 is not critical for the binding of H4B and imply that some of the protein residues involved in dimer formation and in preservation of active site integrity are located, probably at the monomer-monomer interface, in the N-terminal end of the protein.

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.

The stearoyl-acyl carrier protein Δ9 desaturase (Δ9D) uses an oxo-bridged diiron center to catalyze the NAD(P)H– and O2–dependent desaturation of stearoyl-ACP. Δ9D, ribonucleotide reductase, and methane monooxygenase have substantial similarities in their amino acid primary sequences and the physical properties of their diiron centers. These three enzymes also appear to share common features of their reaction cycles, including the binding of O2 to the diferrous state and the subsequent generation of transient diferric-peroxo and diferryl species. In order to investigate the coordination environment of the proposed diferric-peroxo intermediate, we have studied the binding of azide to the diiron center of Δ9D using optical, resonance Raman (RR), and transient kinetic spectroscopic methods. The addition of azide results in the appearance of new absorption bands at 325 nm and 440 nm (kapp≈3.5 s–1 in 0.7 M NaN3, pH 7.8). RR experiments demonstrate the existence of two different adducts: an η1–terminal structure at pH 7.8 (14N3– asymmetric stretch at 2073 cm–1, resolved into two bands with 15N14N2–) and a μ-1,3 bridging structure at pH

Resonance Raman (rRaman) spectroscopy has been used extensively in the studies of the heme chemistry of carbon monoxy adducts. In porphyrin systems, the axial ligand vibrational modes of the CO, ν(Fe−CO) and ν(CO), are enhanced with Soret excitation via an A-term (Franck−Condon) mechanism, but are not expected with Q excitation (B-term or vibronic mechanism). For the first time, these modes have been obtained with Qy as well as Soret excitation in rRaman spectra of CO complexes of ferrous chlorins. The enhancement with Qy excitation arises from an A-term mechanism of Raman scattering for these chlorins owing to their reduced molecular symmetry. Thus, in iron chlorins or other heme systems with reduced molecular symmetry, axial ligand vibrational modes may be enhanced with Qy excitation if they are observed with Soret excitation in the corresponding iron porphyrins. These findings show rRaman spectroscopy to be exceptionally valuable in the study of chlorin chromophores with Qy enhancement using red or ora...

The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM. Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im. The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.

A series of site-directed mutants, E35Q, E39Q, and E35Q-D179N, in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium, was created by overlap extension, using the polymerase chain reaction. The mutant genes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidases (MnPs) were purified and characterized. The molecular masses of the mutant proteins, as well as UV-vis spectral features of their oxidized states, were very similar to those of the wild-type enzyme. Resonance Raman spectral results indicated that the heme environment of the mutant MnP proteins also was similar to that of the wild-type protein. Steady-state kinetic analyses of the E35Q and E39Q mutant MnPs yielded K(m) values for the substrate MnII that were approximately 50-fold greater than the corresponding K(m) value for the wild-type enzyme. Likewise, the kcat values for MnII oxidation were approximately 300-fold lower than that for wild-type MnP. With the E35Q-D179N double mutant, the K(m) value for MnII was approximately 120-fold greater, and the kcat value was approximately 1000-fold less than that for the wild-type MnP1. Transient-state kinetic analysis of the reduction of MnP compound II by MnII allowed the determination of the equilibrium dissociation constants (KD) and first- order rate constants for the mutant proteins. The KD values were approximately 100-fold higher for the single mutants and approximately 200-fold higher for the double mutant, as compared with the wild-type enzyme. The first-order rate constants for the single and double mutants were approximately 200-fold and approximately 4000-fold less, respectively, than that of the wild-type enzyme. In contrast, the K(m) values for H2O2 and the rates of compound I formation were similar for the mutant and wild-type MnPs. The second-order rate constants for p-cresol and ferrocyanide reduction of the mutant compounds II also were similar to those of the wild-type enzyme.

His-25 and His-132 are the primary candidates for the proximal heme iron ligand in heme oxygenase isozyme-1 (HO-1). The unambiguous spectroscopic demonstration that His-25 is the proximal iron ligand leaves the role of His-132 uncertain. Absorption and resonance Raman spectroscopy are used here to establish that mutation of His-132 to an alanine, glycine, or serine does not alter the histidine-iron bond, but results in the loss of the water molecule coordinated to the distal side of the iron in the wild-type enzyme-substrate complex. The His-132 mutations also (a) destabilize the ferrous-O2 complex with respect to autoxidation, which should result in partial uncoupling of NADPH consumption from heme oxidation, and (b) decrease the affinity of the enzyme for heme. The catalytic activity of the protein is decreased but not suppressed by these mutations: the H132G and H132A mutants retain 40-50% and the H132S mutant 20% of the activity of the wild-type protein. His-132, however, is required for catalytic turnover of the protein with H2O2. These results place His-132 close to the iron on the distal side of the heme pocket and indicate that His-132 facilitates, but is not absolutely required for, the catalytic turnover of HO-1.

The binding of ferrous and ferric hemes and manganese(II)- and manganese(III)-substituted hemes to heme oxygenase has been investigated by optical absorption, resonance Raman, and EPR spectroscopy. The results are consistent with the presence of a six-coordinate heme moiety ligated to an essential histidine ligand and a water molecule. The latter ionizes with a pKa approximately 8.0 to give a mixture of high-spin and low-spin six-coordinate hydroxo adducts. Addition of excess cyanide converts the heme to a hexacoordinate low-spin species. The resonance Raman spectrum of the ferrous heme-heme oxygenase complex and that of the Mn(II)protoporphyrin-heme oxygenase complex shows bands at 216 and 212 cm-1, respectively, that are assigned to the metal-histidine stretching mode. The EPR spectrum of the oxidized heme-heme oxygenase complex has a strongly axial signal with g parallel of approximately 6 and g perpendicular approximately 2. 14NO and 15NO adducts of ferrous heme-heme oxygenase exhibit EPR hyperfine splittings of approximately 20 and approximately 25 Gauss, respectively. In addition, both nitrosyl complexes show additional superhyperfine splittings of approximately 7 Gauss from spin-spin interaction with the proximal histidine nitrogen. The heme environment in the heme-heme oxygenase enzyme-substrate complex has spectroscopic properties similar to those of the heme in myoglobin. Hence, there is neither a strongly electron-donating fifth (proximal) ligand nor an electron-withdrawing network on the distal side of the heme moiety comparable to that for cytochromes P-450 and peroxidases. This observation has profound implications about the nature of the oxygen-activating process in the heme-->biliverdin reaction that are discussed in this paper.

The copper center of the Pseudomonas aeruginosa His117Gly azurin mutant is accessible to exogenous ligands through an aperture in its surface created by the removal of the endogenous imidazole ligand. Depending on the exogenous ligand, a surprising variety of type 1 and type 2 copper sites can be obtained that are readily distinguished by electronic, EPR, and resonance Raman (RR) spectroscopy. The RR spectrum of type 1 H117G with exogenous imidazole is nearly identical to that of wild-type azurin, indicating that the trigonal geometry and short Cu-S(Cys) bond of approximately 2.15 A have been maintained. With anionic ligands (e.g., Cl-, Br-, N3-), the RR spectra show increased intensity at 370 and 400 cm-1 and a corresponding decrease in intensity at 410 cm-1, suggesting a lengthening of the Cu-S(Cys) bond as the site achieves a more tetrahedral character. An extreme example is the hydroxide adduct of H117G which is green in color and has optical and RR spectra reminiscent of the tetrahedral type 1 site in Achromobacter cycloclastes nitrite reductase. The fact that the basic RR pattern is little changed in most of the type 1 adducts indicates that the RR spectrum is due primarily to vibrations of the Cu-cysteinate moiety and that its coplanar conformation is conserved. Type 2 H117G proteins are formed by the addition of bidentate exogenous ligands such as histidine and histamine. They have their absorption maxima blue-shifted to 400 nm and their EPR A parallel values increased to approximately 160 x 10(-4) cm-1, both of which are characteristic of tetragonal Cu sites with Cu-S(thiolate) bonds of > 2.25 A. The RR spectra of the type 2 H117G proteins are still dominated by multiple cysteinate-related vibrational modes. However, the vibrational modes with the greatest intensity and Cu-S(Cys) stretching character have shifted approximately 100 cm-1 to lower energy compared to the type 1 sites, consistent with a longer (Cys)S-Cu bond. It is proposed that the tetragonal type 2 character of the bidentate ligand complexes is due to the addition of a fourth strong ligand in the equatorial ligand plane.

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.

Nitrite reductase (NiR) from Achromobacrer cycloclastes and mutant yeast Cu-Zn superoxide dismutase (with Cys substituted for His80 and Cu for Zn) have both been shown to contain type 1 Cu sites. However, they differ from other type 1 (blue) Cu proteins in that they are green: the absorption band at ∼460 nm is more intense than the one at ∼600 nm. Excitation within either of these absorption bands leads to resonance Raman (RR) spectra that are characteristic of type 1 Cu with a large number of peaks between 250 and 500 cm -1 . The RR spectra of NiR and mutant SOD are thus indicative of a Cu-cysteinate chromophore with a short Cu-S bond distance (∼2.1 A) and a coplanar cysteine moiety (Cu-S γ -C β -C α dihedral angle ∼180 o )

Iron-sulfur proteins and cupredoxins are electron-transfer proteins containing metal-cysteinate ligation. Both classes of proteins have intense (Cys)S→M ox charge-transfer bands that are effective for resonance enhancement of Raman-active modes of vibration of the metal ion chromophores. Resonance Raman (RR) spectroscopy provides a sensitive and powerful method for the investigation of the structural details of the metal clusters. The abundance of normal modes in plant ferredoxins and high-potential iron proteins has been shown to arise from coupling of the Fe-S stretching modes with an S−C−C deformation mode that is at a maximum when the Fe−S−C−C dihedral angle is ∼180°

Cyanide has been investigated as a potential ligand-directed probe of the coordination chemistry of Cu(II) and Cu(I) active sites via vibrational spectroscopic studies of CN - coordinated to the metal centers in non-blue copper proteins. Native superoxide dismutase (SOD) was found to bind one CN - that had IR and Raman frequencies at 2137 cm -1

The copper complex [Cu 2 (XYL-O-)(OH)] 2+ , where XYL=m-xylene connected to twobis[2-(2-pyridyl)ethyl]amine ligands, serves as a potential model for the dinuclear copper sites in hemocyanin, tyrosinase, and the multicopper oxidases. Resonance Raman (RR) and infrared spectra of the complex reveal a v s (Cu-OH-Cu) mode at 465 cm -1 and a v as (Cu-OR-Cu) mode at 603 cm -1 . These represent the first such modes to be defitively identified by isotopic shifts and polarization behavior

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.

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 antiferromagnetic coupling between the low-spin FeIII and the superoxide ligand. The Fe-O2 stretching vibration of the oxygenated peroxidase is observed at 563 cm-1 and shifts to 538 cm-1 with 18O isotope. The Fe-histidine stretching vibration is observed at 245 cm-1 in ferrous peroxidase and appears to shift to 276 cm-1 in the oxygenated complex. ? 1990 American Chemical Society.

We report herein the resonance Raman, electronic absorption, and Fourier transform intrared (IR) spectra of tetracoordinate Cu II and Ni II complexes of cis- and trans-octaethylchlorin 23 (Figure 1). Both the stereochemistry at the pyrroline ring and the macrocyclic conformation (planar vs S 4 -ruffled) are clearly shown to influence the spectral properties of the M(OEC) complexes

To evaluate the effects of sequential pyrrole ring reduction, we have examined the vibrational spectra of tetracoordinate Zn(II) complexes of meso-tetraphenylporphyrin [Zn(TPP)], meso-tetraphenylchlorin [Zn(TPC)], and meso-tetraphenylisobacteriochlorin [Zn(TPiBC)]. The phenyl substituents of these macrocycles fall into distinct symmetry classes; for Zn(TPP), all four phenyl groups are equivalent; for Zn(TPC), the phenyl groups are of two different types; and for Zn(TPiBC), there are three types of phenyl substituent

The excited state of Ir 2 (TMB) 4 2+ (TMB=2,5-dusocyano-2,5-dimethylhexane) reacts with hydrogen atom donors to give Ir 2 (TMB) 4 H 2 2+ (Ir 2 H 2 ). This d 7 -d 7 dihydride has been isolated as a tetraphenylborate salt: crystallizes in the monoclinic system, space group P2 1 /c

Reduction of Zr(O-2,6-Me(2)C(6)H(3))Cl(2)[N(SiMe(2)CH(2)PPr(i)(2))(2)] with sodium amalgam under dinitrogen yields the dinuclear zirconium dinitrogen complex {[(Pr(i)(2)PCH(2)SiMe(2))(2)N]Zr(O-2,6-Me(2)C(6)H(3))}(2)(mgr;-eta(2):eta(2)-N(2)). Solid state structural analysis shows that the dinitrogen unit is bound in a side-on mode of coordination with the N-N bond distance at 1.528(7) Å; resonance Raman spectra show a band at 751 cm(-)(1) for nu(N-N), which is consistent with this very long bond. In addition, the N(2) ligand is hinged slightly so that the Zr(2)(mgr;-eta(2):eta(2)-N(2)) core adopts a flattened butterfly shape rather than a completely planar core as found in other related systems. Other Zr(IV) precursors of the general formula ZrCl(2)X[N(SiMe(2)CH(2)PPr(i)(2))(2)] (X = OBu(t), OCHPh(2), NPh(2)) either decompose upon reduction under N(2) or produce mixtures of products.

The outcome of O2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122*) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)2-(carboxylate)4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H(peroxo)) in O2 activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extra" electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the mu-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122* and which converts very slowly (t1/2 approximately 7 h) upon incubation at 0 degrees C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16O2 or 18O2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O2. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mössbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which mu-O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon-hydroxyphenylalanine is formed and pathways leading to Y122* formation predominate in both R2-D84E and R2-W48F.

The inability of imidazole to complement function in the axial histidine deletion mutant, H175G, of yeast cytochrome c peroxidase has been an intriguing but unresolved issue that impacts our understanding of the role of axial ligands in heme catalysis. Here we report the functional and spectroscopic properties of H175G and of its complexes with imidazole. Combined with the crystal structures for these complexes, the data provide a detailed and consistent account of the modes of Im binding in the H175G cavity and their dependence on buffer and pH. UV--vis, EPR, and resonance Raman spectra reveal multiple coordination states for H175G/Im which can be correlated with the crystal structures to assign the following heme environments: H175G/H(2)O/H(2)O, H175G/Im(d)/phosphate(c), H175G/Im(d)/H(2)O(c), H175G/Im(c)/H(2)O(d), and H175G/Im(c)/OH(-)(c), where H175G/X/Y defines the proximal species as X and the distal species as Y and c and d subscripts refer, where known, to the coordinated and dissociated states, respectively. Resonance Raman data for reduced H175G/Im show two substates for heme-coordinated Im differing in the strength of their hydrogen bond to Asp-235, in a fashion similar to WT CCP. NO binding to ferrous H175G/Im results in dissociation of Im from the heme but not from the cavity, while no dissociation is observed for WT CCP, indicating that steric tethering may, in part, control NO-induced dissociation of trans ligands. H175G/Im forms an oxidized compound I state with two distinct radical species, each with a dramatically different anisotropy and spin relaxation from that of the Trp-191 radical of WT CCP. It is suggested that these signals arise from alternate conformations of Trp191 having different degrees of exchange coupling to the ferryl heme, possibly mediated by the conformational heterogeneity of Im within the H175G cavity. The kinetics of the reaction of H175G/Im with H(2)O(2) are multiphasic, also reflecting the multiple coordination states of Im. The rate of the fastest phase is essentially identical to that of WT CCP, indicating that the H175G/Im(c)/H(2)O(d) state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is5% of WT and is unaffected by Im coordination. In summary, Im coordination to H175G results in a number of conformers, one of which is structurally and spectroscopically very similar to WT CCP. However, while this form is fully reactive with peroxide, the reaction with cytochrome c remains inefficient, perhaps implicating the altered Trp-191 radical species.

The H25C and H25Y mutants of human heme oxygenase-1 (hHO-1), in which the proximal iron ligand is replaced by a cysteine or tyrosine, have been expressed and characterized. Resonance Raman studies indicate that the ferric heme complexes of these proteins, like the complex of the H25A mutant but unlike that of the wild type, are 5-coordinate high-spin. Labeling of the iron with 54Fe confirms that the proximal ligand in the ferric H25C protein is a cysteine thiolate. Resonance-enhanced tyrosinate modes in the resonance Raman spectrum of the H25Y.heme complex provide direct evidence for tyrosinate ligation in this protein. The H25C and H25Y heme complexes are reduced to the ferrous state by cytochrome P450 reductase but do not catalyze alpha-meso-hydroxylation of the heme or its conversion to biliverdin. Exposure of the ferrous heme complexes to O2 does not give detectable ferrous-dioxy complexes and leads to the uncoupled reduction of O2 to H2O2. Resonance Raman studies show that the ferrous H25C and H25Y heme complexes are present in both 5-coordinate high-spin and 4-coordinate intermediate-spin configurations. This finding indicates that the proximal cysteine and tyrosine ligand in the ferric H25C and H25Y complexes, respectively, dissociates upon reduction to the ferrous state. This is confirmed by the spectroscopic properties of the ferrous-CO complexes. Reduction potential measurements establish that reduction of the mutants by NADPH-cytochrome P450 reductase, as observed, is thermodynamically allowed. The two proximal ligand mutations thus destabilize the ferrous-dioxy complex and uncouple the reduction of O2 from oxidation of the heme group. The proximal histidine ligand, for geometric or electronic reasons, is specifically required for normal heme oxygenase catalysis.

Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Delta9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous enzyme, followed by O-O bond cleavage and the formation of high-valent iron intermediates [formally Fe(IV)] that are thought to be the reactive oxidants. Greater kinetic stability of a peroxodiiron(III) intermediate in protein R2 of ribonucleotide reductase was achieved by the iron-ligand mutation Asp84 --Glu and the surface mutation Trp48 --Phe. Here, we present the first definitive evidence for a bridging, symmetrical peroxo adduct from vibrational spectroscopic studies of the freeze-trapped intermediate of this mutant R2. Isotope-sensitive bands are observed at 870, 499, and 458 cm-1 that are assigned to the intraligand peroxo stretching frequency and the asymmetric and symmetric Fe-O2-Fe stretching frequencies, respectively. Similar results have been obtained in the resonance Raman spectroscopic study of a peroxodiferric species of Delta9-stearoyl-ACP desaturase [Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry 37, 14664-14671]. Similarities among these adducts and transient species detected during O2 activation by methane monooxygenase hydroxylase, ferritin, and wild-type protein R2 suggest the symmetrical peroxo adduct as a common intermediate in the diverse oxidation reactions mediated by members of this class.

In the redox center of azurin, the Cu(II) is strongly coordinated to one thiolate S from Cys 112 and two imidazole Ns from His 46 and 117. This site yields a complex resonance Raman (RR) spectrum with >20 vibrational modes between 200 and 1500 cm–1. We have investigated the effects of ligand-selective isotope replacements on the RR spectrum of Pseudomonas aeruginosa azurin to determine the relative spectral contribution from each of the copper ligands. Growth on 34S-sulfate labels the cysteine ligand and allows the identification of a cluster of bands with Cu–S(Cys) stretching character between 370 and 430 cm–1 whose frequencies are consistent with the trigonal or distorted tetrahedral coordination in type 1 sites. In type 2 copper-cysteinate sites, the lower ν (Cu–S) frequencies between 260 and 320 cm–1 are consistent with square-planar coordination. Addition of exogenous15N-labeled imidazole or histidine to the His117Gly mutant generates type 1 or type 2 sites, respectively. Because neither the above nor the His46Gly mutant reconstituted with 15N-imidazole exhibits significant isotope dependence, the histidine ligands can be ruled out as important contributors to the RR spectrum. Instead, a variety of evidence, including extensive isotope shifts upon global substitution with 15N, suggests that the multiple RR modes of azurin are due principally to vibrations of the cysteine ligand. These are resonance-enhanced through kinematic coupling with the Cu–S stretch in the ground state or through an excited-state A-term mechanism involving a Cu-cysteinate chromophore that extends into the peptide backbone.

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q.,Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C.,Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J.,Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B.,Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.

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.