Crystal structure of manganese catalase from Lactobacillus plantarum.

BACKGROUND: Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex. RESULTS: The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase. CONCLUSIONS: A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.

Catalytic reaction profile for alcohol oxidation by galactose oxidase.

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme turnover. Recent studies using 1-O-methyl alpha-D-galactopyranoside have revealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha-deuterated alcohol (kH/kD = 22), demonstrating that cleavage of the 6,6'-di[2H]hydroxymethylene C-H bond is fully rate-limiting for oxidation of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for catalysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochemistry 37, 8426-8436]. In the work presented here, the enzyme's unusually broad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of the hydroxyl group undergoing oxidation. Quantitative structure-activity relationship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for these reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relatively difficult to oxidize nitro derivative, the KIE is large (kH/kD = 12.3), implying rate-limiting C-H bond cleavage for the oxidation reaction. This contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (kH/kD = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-nitrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (kH2O/kD2O = 1.2). These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen atom transfer) comprising a single-step proton-coupled electron transfer mechanism. Each component exhibits a distinct substituent and isotope sensitivity, allowing them to be identified kinetically. The proton transfer component is unique in being sensitive to the isotopic character of the solvent (H2O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstraction from a coordinated substrate activates the alcohol toward inner sphere electron transfer to the Cu(II) metal center in an unfavorable redox equilibrium, forming an alkoxy radical which undergoes hydrogen atom abstraction by the tyrosine-cysteine phenoxyl free radical ligand to form the product aldehyde.

Removing a hydrogen bond in the dimer interface of Escherichia coli manganese superoxide dismutase alters structure and reactivity.

Among manganese superoxide dismutases, residues His30 and Tyr174 are highly conserved, forming part of the substrate access funnel in the active site. These two residues are structurally linked by a strong hydrogen bond between His30 NE2 from one subunit and Tyr174 OH from the other subunit of the dimer, forming an important element that bridges the dimer interface. Mutation of either His30 or Tyr174 in Escherichia coli MnSOD reduces the superoxide dismutase activity to 30--40% of that of the wt enzyme, which is surprising, since Y174 is quite remote from the active site metal center. The 2.2 A resolution X-ray structure of H30A-MnSOD shows that removing the Tyr174-->His30 hydrogen bond from the acceptor side results in a significant displacement of the main-chain segment containing the Y174 residue, with local rearrangement of the protein. The 1.35 A resolution structure of Y174F-MnSOD shows that disruption of the same hydrogen bond from the donor side has much greater consequences, with reorientation of F174 having a domino effect on the neighboring residues, resulting in a major rearrangement of the dimer interface and flipping of the His30 ring. Spectroscopic studies on H30A, H30N, and Y174F mutants show that (like the previously characterized Y34F mutant of E. coli MnSOD) all lack the high pH transition of the wt enzyme. This observation supports assignment of the pH sensitivity of MnSOD to coordination of hydroxide ion at high pH rather than to ionization of the phenolic group of Y34. Thus, mutations near the active site, as in the Y34F mutant, as well as at remote positions, as in Y174F, similarly affect the metal reactivity and alter the effective pK(a) for hydroxide ion binding. These results imply that hydrogen bonding of the H30 imidazole N--H group plays a key role in substrate binding and catalysis.

Outer sphere mutations perturb metal reactivity in manganese superoxide dismutase.

Tyrosine 34 and glutamine 146 are highly conserved outer sphere residues in the mononuclear manganese active site of Escherichia coli manganese superoxide dismutase. Biochemical and spectroscopic characterization of site-directed mutants has allowed functional characterization of these residues in the wild-type (wt) enzyme. X-ray crystallographic analysis of three mutants (Y34F, Q146L, and Q146H) reveal subtle changes in the protein structures. The Y34A mutant, as well as the previously reported Y34F mutant, retained essentially the full superoxide dismutase activity of the wild-type enzyme, and the X-ray crystal structure of Y34F manganese superoxide dismutase shows that mutation of this strictly conserved residue has only minor effects on the positions of active site residues and the organized water in the substrate access funnel. Mutation of the outer sphere solvent pocket residue Q146 has more dramatic effects. The Q146E mutant is isolated as an apoprotein lacking dismutase activity. Q146L and Q146H mutants retain only 5-10% of the dismutase activity of the wild-type enzyme. The absorption and circular dichroism spectra of the Q146H mutant resemble corresponding data for the superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilum, which is active in both Mn and Fe forms. Interestingly, the iron-substituted Q146H protein also exhibits low dismutase activity, which increases at lower pH. Mutation of glutamine 146 disrupts the hydrogen-bonding network in the active site and has a greater effect on protein structure than does the Y34F mutant, with rearrangement of the tyrosine 34 and tryptophan 128 side chains.

Expression of recombinant galactose oxidase by Pichia pastoris.

Galactose oxidase catalyzes the oxidation of a variety of primary alcohols, producing hydrogen peroxide as a product. Among hexose sugars, the enzyme exhibits a high degree of specificity for the C6-hydroxyl of galactose and its derivatives, underlying a number of important bioanalytical applications. Galactose oxidase cDNA has been cloned for expression in Pichia pastoris both as the full-length native sequence and as a fusion with the glucoamylase signal peptide. Expression of the full-length native sequence results in a mixture of partly processed and mature galactose oxidase. In contrast, the fusion construct directs efficient secretion of correctly processed galactose oxidase in high-density, methanol-induced fermentation. Culture conditions (including induction temperature and pH) have been optimized to improve the quality and yield (500 mg/L) of recombinant enzyme. Lowering the temperature from 30 to 25 degrees C during the methanol induction phase results in a fourfold increase in yield. A simple two-step purification and one-step activation produce highly active galactose oxidase suitable for a wide range of biomedical and bioanalytical applications.

Recombinant superoxide dismutase from a hyperthermophilic archaeon, Pyrobaculum aerophilium.

Superoxide dismutase (SOD) from the hyperthermophilic archaeon Pyrobaculum aerophilum (a facultative aerobe) has been cloned and expressed in a mesophilic host (Escherichia coli) as a soluble tetrameric apoprotein. The purified apoprotein can be reconstituted with either Mn or Fe by heating the protein with the appropriate metal salt at an elevated temperature (95 degrees C). Both Mn- and Fe-reconstituted P. aerophilum SOD exhibit superoxide dismutase activity, with the Mn-containing enzyme having the higher activity. P. aerophilum SOD is extremely thermostable and the reconstitution with Mn(II) can be performed in an autoclave (122 degrees C, 18 psi). The Mn(III) optical absorption spectrum of Mn-reconstituted P. aerophilum SOD is distinct from that of most other MnSODs and is unchanged upon addition of NaN3. The optical absorption spectrum of Fe-reconstituted P. aerophilum SOD is typical of Fe-substituted MnSODs and authentic FeSOD and exhibits a pH-dependent transition with an effective pKa value higher than that found for Fe-substituted MnSOD from either E. coli or Thermus spp. Amino acid sequence analysis shows that the P. aerophilum SOD is closely related to SODs from other hyperthermophilic archaea (Aeropyrum pernix and Sulfolobus spp.), forming a family of enzymes distinct from the hyperthermophilic bacterial SOD from Aquifex pyrophilus and from mesophilic SODs.

Identification of catalytic residues in glyoxal oxidase by targeted mutagenesis.

Glyoxal oxidase is a copper metalloenzyme produced by the wood-rot fungus Phanerochaete chrysosporium as an essential component of its extracellular lignin degradation pathways. Previous spectroscopic studies on glyoxal oxidase have demonstrated that it contains a free radical-coupled copper active site remarkably similar to that found in another fungal metalloenzyme, galactose oxidase. Alignment of primary structures has allowed four catalytic residues of glyoxal oxidase to be targeted for site-directed mutagenesis in the recombinant protein. Three glyoxal oxidase mutants have been heterologously expressed in both a filamentous fungus (Aspergillus nidulans) and in a methylotrophic yeast (Pichia pastoris), the latter expression system producing as much as 2 g of protein per liter of culture medium under conditions of high density methanol-induced fermentation. Biochemical and spectroscopic characterization of the mutant enzymes supports structural correlations between galactose oxidase and glyoxal oxidase, clearly identifying the catalytically important residues in glyoxal oxidase and demonstrating the functions of each of these residues.

Thermally triggered metal binding by recombinant Thermus thermophilus manganese superoxide dismutase, expressed as the apo-enzyme.

Manganese superoxide dismutase from the extremely thermophilic eubacterium Thermus thermophilus has been cloned and expressed at high levels in a mesophilic host (Escherichia coli) as a soluble tetrameric protein mainly present as the metal-free apo-enzyme. Incubation of the purified apo-enzyme with manganese salts at ambient temperature did not restore superoxide dismutase activity, but reactivation could be achieved by heating the protein with Mn(II) at higher temperatures, approaching the physiological growth temperature for T. thermophilus. Heat annealing followed by incubation with manganese at lower temperature fails to reactivate the enzyme, demonstrating that a simple misfolding of the protein is not responsible for the observed behavior. The in vitro metal uptake is nonspecific, and manganese, iron, and vanadium all bind, but only manganese restores catalytic activity. Bound metal ions do not exchange during heat treatment, indicating that the formation of the metal complex is effectively irreversible under these conditions. The metallation process is strongly temperature-dependent, suggesting that substantial activation barriers to metal uptake at ambient temperature are overcome by a thermal transition in the apo-protein structure. A mechanism for SOD metallation is proposed, focusing on interactions at the domain interface.

The oxidized (3,3) state of manganese catalase. Comparison of enzymes from Thermus thermophilus and Lactobacillus plantarum.

Manganese catalases contain a binuclear manganese cluster that catalyzes the redox dismutation of hydrogen peroxide, interconverting between dimanganese(II) [(2,2)] and dimanganese(III) [(3,3)] oxidation states during turnover. We have investigated the oxidized (3,3) states of the homologous enzymes from Thermus thermophilus and Lactobacillus plantarum using a combination of optical absorption, CD, MCD, and EPR spectroscopies as sensitive probes of the electronic structure and protein environment for the active site metal clusters. Comparison of results for these two enzymes allows the essential features of the active sites to be recognized and the differences identified. For both enzymes, preparations having the highest catalytic activity have diamagnetic ground states, consistent with the bis-mu-bridging dimanganese core structure that has been defined crystallographically. Oxidative damage and exogenous ligand binding perturb the core structure of LPC, converting the enzyme to a distinct form in which the cluster becomes paramagnetic as a result of altered exchange coupling mediated by the bridging ligands. The TTC cluster does not exhibit this sensitivity to ligand binding, implying a different reactivity for the bridges in that enzyme. A mechanism is proposed involving distinct coordination modes for peroxide substrate in each of the two half-reactions for enzyme turnover.

A glutamate bridge is essential for dimer stability and metal selectivity in manganese superoxide dismutase.

In Escherichia coli manganese superoxide dismutase (MnSOD), the absolutely conserved Glu170 of one monomer is hydrogen-bonded to the Mn ligand His171 of the other monomer, forming a double bridge at the dimer interface. Point mutation of Glu170 --> Ala destabilizes the dimer structure, and the mutant protein occurs as a mixture of dimer and monomer species. The purified E170A MnSOD contains exclusively Fe and is devoid of superoxide dismutase activity. E170A Fe2-MnSOD closely resembles authentic FeSOD in terms of spectroscopic properties, anion interactions and pH titration behavior. Reconstitution of E170A Fe2-MnSOD with Mn(II) salts does not restore superoxide dismutase activity despite the spectroscopic similarity between E170A Mn2-MnSOD and wild type Mn2-MnSOD. Growth of sodA+ and sodA- E. coli containing the mutant plasmid pDT1-5(E170A) is impaired, suggesting that expression of mutant protein is toxic to the host cells.

Kinetic isotope effects as probes of the mechanism of galactose oxidase.

Galactose oxidase (GO) is a member of the family of radical-coupled copper oxidases, enzymes containing a free radical coordinated to copper in the active site. In catalysis GO cycles between an oxidized state (comprising Cu(II) with a unique cysteinyl-tyrosine radical) and a reduced state (comprising Cu(I) with the singlet cysteinyl-tyrosine) as it catalyzes the two-electron oxidation of alcohols to aldehydes and the subsequent reduction of O2 to H2O2. A ping-pong mechanism involving radical intermediates has been proposed for GO catalysis. Previous steady-state kinetics studies have demonstrated a KIE of 7-8 that was attributed to substrate oxidation, a process involving the stereospecific abstraction of the pro-S hydrogen from the 6-hydroxymethyl group of galactose. We have used rapid kinetics methods to measure the anaerobic reduction of GO substrate at 4 degreesC and carry out enzyme-monitored turnover experiments using 6-protio and 6-deutero substrates, both in H2O and D2O. At concentrations below Km, the apparent second-order rate constant for protio-substrate oxidation, kred, was 1.59 x 10(4) M-1 s-1, while that for deuterated substrate was 7.50 x 10(2) M-1 s-1, a KIE of 21.2. Steady-state measurements of oxygen consumption at low galactose concentrations reveal an unusually large isotope effect (kH/kD = 22.5 +/- 2) for oxidation of 1-O-methyl-6, 6'-di-[2H]-alpha-d-galactopyranoside, and at high galactose concentrations, where the oxygen half-reaction is rate-limiting in catalysis, a surprisingly large KIE (kH/kD = 8 +/- 1) for the reduction of O2 to H2O2. There is no detectable solvent isotope effect (<5%) on any of these measurements. This shows that there are no exchangeable protons involved in any kinetically significant step and that the hydrogen atom removed from galactose is not lost to solvent during catalysis; instead, it also participates in the rate-limiting step of the subsequent reaction with oxygen. At concentrations below Km, apparent second-order rate constants for protio-substrate oxidation (kred = 1.5 x 10(4) M-1 s-1) and O2 reduction (kox = 8 x 10(6) M-1 s-1) have been estimated from measurements both by steady-state oxygen electrode and by enzyme-monitored turnover. This is completely consistent with the anaerobic studies mentioned above. Our results show that the enzyme is essentially fully oxidized while in steady-state turnover, consistent with the reduction step being nearly fully rate-limiting at practical substrate concentrations, due to the very fast reaction with physiological concentrations of O2. Overall, the catalytic reaction is in concordance with a ping-pong mechanism. The large KIE associated with reduction of the enzyme in all three methods appears to reflect hydrogen atom radical abstraction by the active site tyrosine radical in the rate-determining step, in agreement with the previously proposed radical mechanism for GO. The KIE determined at low substrate concentrations (where oxidation of substrate is rate determining) from steady-state oxygen consumption measurements, varies from 22.5 at 4 degreesC to 13 at 45 degreesC, consistent with tunneling being involved in the hydrogen atom transfer step.

Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase.

Mutagenesis of Escherichia coli manganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions. Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six-coordinate azide complex. Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands. However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands. In vivo substitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme. This pH-coupled transition shifts once more to a higher effective pKa for Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme. Peroxide sensitivities of the Fe complexes are distinct for the wild-type and mutant proteins, indicating a role for Y34 in peroxide interactions. These results provide evidence for a conserved peroxide-protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands.

Low-temperature thermochromism marks a change in coordination for the metal ion in manganese superoxide dismutase.

We have observed thermochromism (temperature-dependent absorption) for anion complexes of manganese superoxide dismutase indicating a change in coordination number for the metal complex at low temperatures. The ligand field spectra for the Mn(III) ion, characteristic of five-coordination for the azide complex at 295 K, cleanly convert to spectra reflecting six-coordination at low temperature, with a midpoint for the transition near 200 K. The active site structure is temperature-dependent, a relatively rigid, distorted octahedral low-temperature Mn complex melting with dehydration (or displacement of one of the protein ligands) to form a five-coordinated complex under physiological conditions. Thermodynamic parameters for the transition estimated from van't Hoff analysis (delta HvH = 5 kcal/mol; delta SvH = 22 cal/mol K) are consistent with reduced chemical binding and increased fluxionality at room temperature. This thermochromism of MnSD demonstrates the existence of distinct isomeric forms of the active site metal complex, whose relative stability depends on the degree of vibrational excitation. The marginal destabilization of the six-coordinate anion complex under physiological conditions suggests that the enzyme may thermally control the stability of intermediates in a dissociative displacement mechanism for substrate binding and redox.

EPR polarization studies on Mn catalase from Lactobacillus plantarum.

The binuclear manganese active site of Mn catalase catalyzes redox disproportionation of hydrogen peroxide, forming dioxygen and water. We report here multifrequency EPR and microwave polarization studies of the catalytically active homovalent Mn2+ complex of Lactobacillus plantarum Mn catalase, resolving spectra from each of the thermally accessible multiplet states of the coupled complex by multivariate methods. The experimental spectra have been simulated using computational approaches for the binuclear cluster to predict both intensity and polarization for arbitrary values of the ground state parameters. These two spectroscopic properties define the nature of the ground state wavefunctions and so serve as a sensitive and quantitative measure of the inter-ion interactions in the reduced complex. Interpretation of the spectra in terms of a pair Hamiltonian that includes Heisenberg exchange, dipolar, single site zero field splitting, and Zeeman perturbations leads to the most complete ground state description of the active site metal centers. The results of this spectroscopic analysis support a picture of two high spin ions weakly coupled by exchange interactions (J = 40 cm-1) with relatively small dipole-dipole coupling and single site zero field splittings for the ligand-free reduced enzyme. The coupling between fluoride binding and protonation of the complex has been demonstrated by proton uptake studies. The binding of two fluoride ions in the active site dramatically changes the pair spectra, reflecting a substantially reduced J-coupling (J = 10.5 cm-1) that must be a consequence of perturbation of the bridging ligands. Anion binding to the binuclear Mn complex appears to result in poisoning of the active site by protons, possibly associated with insertion of fluoride into bridging positions of the dimanganese core.

Glyoxal oxidase from Phanerochaete chrysosporium is a new radical-copper oxidase.

A free radical-coupled copper complex has been identified as the catalytic structure in the active site of glyoxal oxidase from Phanerochaete chrysosporium based on a combination of spectroscopic and biochemical studies. The native (inactive) enzyme is activated by oxidants leading to the elimination of the cupric EPR signal consistent with formation of an antiferromagnetically coupled radical-copper complex. Oxidation also leads to the appearance of a substoichiometric free radical EPR signal with an average g value (gav = 2.0055) characteristic of phenoxyl tau-radicals arising from a minority apoenzyme fraction. Optical absorption, CD, and spectroelectrochemical measurements on the active enzyme reveal complex spectra extending into the near IR and define the redox potential for radical formation (E 1/2 = 0.64 V versus NHE, pH 7.0). Resonance Raman spectra have identified the signature of a modified (cysteinyl-tyrosine) phenoxyl in the vibrational spectra of the active complex. This radical-copper motif has previously been found only in galactose oxidase, with which glyoxal oxidase shares many properties despite lacking obvious sequence identity, and catalyzing a distinct reaction. The enzymes thus represent members of a growing class of free radical metalloenzymes based on the radical-copper catalytic motif and appear to represent functional variants that have evolved to distinct catalytic roles.

Ligand interactions with galactose oxidase: mechanistic insights.

Interactions between galactose oxidase and small molecules have been explored using a combination of optical absorption, circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to detect complex formation and characterize the products. Anions bind directly to the cupric center in both active and inactive galactose oxidase, converting to complexes with optical and EPR spectra that are distinctly different from those of the starting aquo enzyme. Azide binding is coupled to stoichiometric proton uptake by the enzyme, reflecting the generation of a strong base (pKa > 9) in the active site anion adduct. At low temperature, the aquo enzyme converts to a form that exhibits the characteristic optical and EPR spectra of an anion complex, apparently reflecting deprotonation of the coordinated water. Anion binding results in a loss of the optical transition arising from coordinated tyrosine, implying displacement of the axial tyrosine ligand on forming the adduct. Nitric oxide binds to galactose oxidase, forming a specific complex exhibiting an unusual EPR spectrum with all g values below 2. The absence of Cu splitting in this spectrum and the observation that the cupric EPR signal from the active site metal ion is not significantly decreased in the complex suggest a nonmetal interaction site for NO in galactose oxidase. These results have been interpreted in terms of a mechanistic scheme where substrate binding displaces a tyrosinate ligand from the active site cupric ion, generating a base that may serve to deprotonate the coordinated hydroxyl group of the substrate, activating it for oxidation. The protein-NO interactions may probe a nonmetal O2 binding site in this enzyme.

A tyrosine-derived free radical in apogalactose oxidase.

Oxidation of apogalactose oxidase with ferricyanide leads to the formation of a stable free radical exhibiting distinctive optical absorption and EPR spectral features. The radical is associated with absorption in both near-UV and near-IR spectral regions, and its EPR spectrum is characteristic of an aromatic free radical with gav = 2.005. Reconstitution of both the apoenzyme and the free radical-containing form with copper substantially restores both the absorption spectra and the catalytic activity of the active enzyme, indicating that the preparation of the radical species does not significantly damage the protein. The absence of a free radical EPR signal in reconstituted and activated galactose oxidase containing nearly stoichiometric copper suggests the radical is an active site species relating to the free radical-coupled copper site previously proposed for this enzyme. Isotopic labeling experiments demonstrate that the radical derives from a tyrosine residue. The distinctive spectra associated with this radical indicate an environment which is different from that associated with the tyrosyl phenoxyl sites in other free radical enzymes.

Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase.

Resonance Raman data are reported for the redox-activated form of galactose oxidase from Dactylium dendroides. Excitation within the red (659 nm) and blue (457.9 nm) absorption bands leads to strong resonance enhancement of ligated tyrosine vibrational modes at 550, 1170, 1247, 1484, and 1595 cm-1. The ring mode frequencies are unusually low, indicating a decreased bond order in the ring. The spectra clearly differ in both frequencies and relative intensities from those characteristic of known aromatic pi-radicals. Enhancement of tyrosine ring modes on excitation within absorption bands previously associated with the presence of the radical in the active site suggests that the ligated tyrosine residue is present in the radical site and may stabilize this radical species through formation of a charge transfer complex. A dramatically different Raman spectrum is observed for the N3- adduct of galactose oxidase, exhibiting a single strong 1483 cm-1 feature. The intense visible-near IR absorption bands for galactose oxidase may derive from transitions within a charge transfer complex between an aromatic free radical and a tyrosine-copper complex.

Sequences of 12 monoclonal anti-dinitrophenyl spin-label antibodies for NMR studies.

Eleven monoclonal antibodies specific for a spin-labeled dinitrophenyl hapten (DNP-SL) have been produced for use in NMR studies. They have been named AN01 and AN03-AN12. The stability constants for the association of these antibodies with DNP-SL and related haptens were measured by fluorescence quenching and ranged from 5.0 X 10(4) M-1 to greater than 1.0 X 10(8) M-1. cDNA clones coding for the heavy and light chains of each antibody and of an additional anti-DNP-SL monoclonal antibody, AN02, have been isolated. The nucleic acid sequence of the 5' end of each clone has been determined, and the amino acid sequence of the variable regions of each antibody has been deduced from the cDNA sequence. The sequences are relatively heterogeneous, but both the heavy and the light chains of AN01 and AN03 are derived from the same variable-region gene families as those of the AN02 antibody. AN07 has a heavy chain that is related to that of AN02, and AN09 has a related light chain. AN05 and AN06 are unrelated to AN02 but share virtually identical heavy and light chains. Preliminary NMR difference spectra comparing related antibodies show that sequence-specific assignment of resonances is possible. Such spectra also provide a measure of structural relatedness.

The active site of galactose oxidase.

The copper enzyme galactose oxidase has been prepared in three distinct redox modifications; two of these represent nearly homogeneous preparations of active and inactive species which have been described previously, while the third has never before been reported. Preparation of these redox modifications as homogeneous species has permitted detailed spectroscopic and catalytic studies of each for the first time. We find that the form which has been extensively probed by EPR spectroscopy is devoid of catalytic activity and does not interact with substrate. The detailed characterization of oxidatively activated galactose oxidase and its anion interactions has led to a spectroscopic assignment of the copper oxidation state in this complex which indicates that the one-electron redox process which converts the inactive form to catalytically active enzyme is associated with oxidation of the protein rather than the metal center as has been proposed previously. This oxidation step is required for catalytic activity and is the basis of the two-electron redox reactivity for the enzyme active site: anaerobic addition of hydroxylic substrates results in reduction of the two-electron redox unit, and the spectral features associated with both the copper ion and the non-metal redox center are eliminated, apparently forming a cuprous site. The two-electron reactivity resulting from protein participation in redox catalysis has important implications in this and other mechanisms where oxygen reduction occurs at a mononuclear metal ion active site.