Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase.

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.

Site-directed mutation of the highly conserved region near the Q-loop of the cytochrome bd quinol oxidase from Escherichia coli specifically perturbs heme b595.

Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The dioxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b(595), which is a high-spin heme whose function is not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B. (1999) Biochim. Biophys Acta 1410, 32--50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved residues) predicted to be on the periplasmic side of the membrane between transmembrane helices 8 and 9 in subunit I. The functional importance of this region is investigated in the current work by site-directed mutagenesis. Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) resulted in a catalytically inactive enzyme with abnormal UV--vis spectra. E445A was selected for detailed analysis because of the absence of the absorption bands from heme b(595). Detailed spectroscopic and chemical analyses, indeed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b(595), appears relatively unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b(595).

Roles of the proximal heme thiolate ligand in cytochrome p450(cam).

To examine the roles of the proximal thiolate iron ligand, the C357H mutant of P450(cam) (CYP101) was characterized by resonance Raman, UV, circular dichroism, and activity measurements. The C357H mutant must be reconstituted with hemin for activity to be observed. The reconstituted enzyme is a mixture of high and low spin species. Low temperature (10 degrees C), low enzyme concentration (1 microM), high camphor concentration (1 mM), and 5--50 mM buffer concentrations increase the high to low spin ratio, but under no conditions examined was the protein more than 60% high spin. The C357H mutant has a poorer K(m) for camphor (23 vs 2 microM) and a poorer K(d) for putidaredoxin (50 vs 20 microM) than wild-type P450(cam). The mutant also exhibits a greatly decreased camphor oxidation rate, elevated uncoupling rate, and much greater peroxidase activity. Electron transfer from putidaredoxin to the mutant is much slower than to the wild-type even though redox potential measurements show that the electron transfer remains thermodynamically favored. These experiments confirm that the thiolate ligand facilitates the O--O bond cleavage by P450 enzymes and also demonstrate that this ligand satisfies important roles in protein folding, substrate binding, and electron transfer.

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function.

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 is <5% 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.

Disruption of an active site hydrogen bond converts human heme oxygenase-1 into a peroxidase.

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 (<3%) of biliverdin forming activity, but the D140F and D140L mutants are inactive in this respect. However, the two latter mutants retain a low ability to form verdoheme, an intermediate in the reaction sequence. All the Asp-140 mutants exhibit a new peroxidase activity. The results indicate that disruption of the distal hydrogen bonding environment by mutation of Asp-140 destabilizes the ferrous dioxygen complex and promotes conversion of the ferrous hydroperoxy intermediate obtained by reduction of the ferrous dioxygen complex to a ferryl species at the expense of its normal reaction with the porphyrin ring.

Resonance Raman studies of the stoichiometric catalytic turnover of a substrate-stearoyl-acyl carrier protein delta(9) desaturase complex.

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.

Formation of a bis(histidyl) heme iron complex in manganese peroxidase at high pH and restoration of the native enzyme structure by calcium.

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.

Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase.

The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H(2)O(2)-dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity.

The active site of the thermophilic CYP119 from Sulfolobus solfataricus.

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.

Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine.

The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.

Arginine 177 is involved in Mn(II) binding by manganese peroxidase.

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were approximately 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H(2)O(2) and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were approximately 10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were approximately 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.

The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins.

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.

Replacement of the proximal histidine iron ligand by a cysteine or tyrosine converts heme oxygenase to an oxidase.

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.

2-Chloro-1,4-dimethoxybenzene cation radical: formation and role in the lignin peroxidase oxidation of anisyl alcohol.

2-Chloro-1,4-dimethoxybenzene (2Cl-1,4-DMB) oxidation by lignin peroxidase (LiP) proceeds via the formation of the 2Cl-1,4-DMB cation radical as indicated by ESR and UV/vis spectroscopy. The products of the LiP-catalyzed oxidation of 2Cl-1,4-DMB were identified as 2-chloro-1,4-benzoquinone and the dimers dichlorotetramethoxybiphenyl and chloro(chlorodimethoxyphenyl)benzoquinone. The addition of anisyl alcohol (AA) rapidly quenched the 2Cl-1,4-DMB cation radical optical absorption bands, suggesting that the cation radical directly mediates the oxidation of AA. When LiP reactions are conducted in the presence of 50 microM 2Cl-1,4-DMB, the enzyme is inactivated; however, this inactivation can be prevented by the addition of AA. This also suggests that the 2Cl-1,4-DMB cation radical formed in the reaction, in turn, oxidizes AA.

O2 activation by non-heme diiron proteins: identification of a symmetric mu-1,2-peroxide in a mutant of ribonucleotide reductase.

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.

Topaquinone-dependent amine oxidases: identification of reaction intermediates by Raman spectroscopy.

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].

Endothelial nitric oxide synthase: modulations of the distal heme site produced by progressive N-terminal deletions.

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.

Heme oxygenase-1, intermediates in verdoheme formation and the requirement for reduction equivalents.

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.

Photoaccumulation in photosystem I does produce a phylloquinone (A1.-) radical.

Previous work has challenged the assignment of a photoaccumulated EPR signal to the phylloquinone electron acceptor in photosystem I, A1.-. Biosynthetic deuteration of the phylloquinone in the cyanobacterium Anabaena variabilis has been shown to narrow this photoaccumulated signal, demonstrating that the signal arises from A1.-. The ESP signal attributed to P700.+A1.- is also narrowed by this deuteration, showing that the photoaccumulated EPR signal and the ESP signal are monitoring the same redox component. Confirmation that the photoaccumulated EPR signal comes from deuterated phylloquinone was obtained by exchanging the deuterated for protonated phylloquinone, which broadened the photoaccumulated EPR signal.