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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.

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.

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.

The crystal structure of nitrous oxide reductase, the enzyme catalyzing the final step of bacterial denitrification in which nitrous oxide is reduced to dinitrogen, exhibits a novel catalytic site, called Cu(Z). This comprises a cluster of four copper ions bound by seven histidines and three other ligands modeled in the X-ray structure as OH(-) or H(2)O. However, elemental analyses and resonance Raman spectroscopy of isotopically labeled enzyme conclusively demonstrate that Cu(Z) has one acid-labile sulfur ligand. Thus, nitrous oxide reductase contains the first reported biological copper-sulfide cluster.

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.

A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.

Hemerythrin (Hr) is an O(2)-carrying protein found in some marine invertebrates. A conserved sequence motif in all Hrs provides five histidine and two carboxylate ligands to an oxo-/hydroxo-bridged diiron active site, as well as a hydrophobic O(2) binding pocket. Database searches located a previously unrecognized Hr-like sequence motif at the 3' end of the gene, dcrH, from the anaerobic sulfate-reducing bacterium, Desulfovibrio (D.) vulgaris (Hildenborough). This gene encodes a putative methyl-accepting chemotaxis protein, DcrH. We have established by immunoblotting that a full-length DcrH, including the Hr-like domain, is expressed in D. vulgaris (Hildenborough). The C-terminal domain of DcrH, when expressed separately in recombinant form in Escherichia coli, was found to fold into a stable protein, DcrH-Hr. The UV-vis absorption and resonance Raman spectra of DcrH-Hr, and of its azide adduct, provide clear evidence for an oxo-bridged diiron(III) site very similar to that found in Hr. Based on UV-vis absorption spectra, exposure of the reduced (colorless, presumably diferrous) DcrH-Hr to air resulted in formation of an O(2) adduct also very similar to that of Hr. Unlike that of Hr, the O(2) adduct of DcrH-Hr autoxidized within a few minutes at room temperature. The O(2) binding pocket of DcrH-Hr appears to be larger than that of Hr. Given the air-sensitive nature of D. vulgaris and the putative chemotactic function of DcrH, one possible role for the Hr-like domain of DcrH is O(2)-sensing. DcrH-Hr is the first characterized example of a Hr-like protein from any microorganism.

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.

Resonance Raman (RR) spectroscopy has proven to be an excellent technique for providing structural information about the 2,4, 5-trihydroxyphenylalaninequinone (TPQ) cofactor and for identifying the source of oxygen atoms during the posttranslational synthesis of the cofactor. Through specific labeling of the C2, C4, and C5 oxygens of TPQ in phenylethylamine oxidase (PEAO) from Arthrobacter globiformis, we have identified the C=O stretch of the C5 carbonyl at 1683 cm-1 (-27 in 18O) and the C=O stretch of the C2 carbonyl at 1575 cm-1 (-21 in 18O). These vibrational frequencies show that the C-O moiety at C5 has far greater double-bond character than at C2 or C4, thereby explaining the exclusive nucleophilic attack at the C5 position by substrates and substrate analogs. Bovine serum amine oxidase (BSAO) exhibits a similar nu(C=O) mode at 1678 cm-1 (-22 cm-1 in 18O). Aniline reacts with the TPQ cofactor of PEAO to form a new derivative (lambdamax at 450 nm) with properties similar to the proposed substrate-imine intermediate in the catalytic cycle. It retains the C2=O spectral features of the native enzyme and exhibits a new C5=N stretch at 1603 cm-1 (-29 in 15N). In contrast, methylamine reacts with both PEAO and BSAO under anaerobic conditions to form a different stable adduct (lambdamax at 385 nm) with properties closer to the proposed product-imine intermediate in the catalytic cycle. This species has a distinctive RR spectrum with a C=N stretch at 1617 cm-1 that corresponds to the atoms of the added methylamine (-58 cm-1 with CD3NH2, -19 cm-1 with CH315NH2). The lack of D2O dependence of nu(C=N) shows that this is a deprotonated imine, which would be more stable toward hydrolysis than the postulated protonated imine in the enzymatic reaction. However, the BSAO product imine (from methylamine) does undergo hydrolysis and conversion to semiquinone upon addition of cyanide. It is possible that the inactive form of the product imine is stabilized by deprotonation and flipping of the TPQ ring [Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J. P. (1997) Biochemistry 36, 11472-11478].

The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.

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.

The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.

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 nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHox) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHox as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHox and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHox for the substrate CH3NH3+, the substrate analogues (CH3)2NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHox in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ.

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.

Combined optical and resonance Raman studies have revealed the formation of an O2-adduct upon exposure of 4e- chemically reduced stearoyl-acyl carrier protein Delta9 desaturase to stearoyl-ACP and 1 atm O2. The observed intermediate has a broad absorption band at 700 nm and is remarkably stable at room temperature (t1/2 approximately 26 min). Resonance Raman studies using 16O2 gas reveal vibrational features of a bound peroxide [Vs(Fe-O2), 442 cm-1; Vas(Fe-O2), 490 cm-1; V(O-O), 898 cm-1] that undergo the expected mass-dependent shifts when prepared in (16)O(18)O or 18(O2). The appearance of two Fe-O2 vibrations, each having a single peak of intermediate frequency with 16(O)18(O), provs that the peroxide is bound symmetrically between the two iron atoms in a mu-1,2 configuration. The same results have been obtained in the accompanying resonance Raman study of ribonucleotide reductase isoform W48F/D84E [P. Moënne-Loccoz, J. Baldwin, B. A. Ley, T. M. Loehr, and J. M. Bollinger, Jr. (1998) Biochemistry 37, 14659-14663], thus making it likely that other members of the class II diiron enzymes form related peroxodiferric intermediates. Study of the reactivity of peroxodiferric Delta9D revealed that this intermediate underwent 2e- reduction leading to an oxidase reaction and recovery of the resting ferric homodimer. In contrast, biological reduction of the same enzyme preparations using ferredoxin reductase and [2Fe-2S] ferredoxin gave catalytic desaturation with a turnover number of 20-30 min-1. The profound difference in catalytic outcome for chemically and enzymatically reduced Delta9D suggests that redox-state dependent conformational changes cause partition of reactivity between desaturase and oxidase chemistries. The Delta9D oxidase reaction represents a new type of reactivity for the acyl-ACP desaturases and provides a two-step catalytic precedent for the "alternative oxidase" activity recently proposed for a membrane diiron enzyme in plants and trypanosomes.