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.

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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.

Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.

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 (g av ‫؍‬ 2.0055) characteristic of phenoxyl -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.The white-rot wood-metabolizing basidiomycete fungi are major degraders of lignin contributing essential chemistry to the global carbon cycle. Phanerochaete chrysosporium, the organism most extensively studied for its lignin-degrading ability, produces three classes of extracellular enzyme under ligninolytic conditions: lignin peroxidase, manganese peroxidase, and glyoxal oxidase (1, 2). In the presence of H 2 O 2 (3), lignin peroxidases oxidize and partially depolymerize lignin or lignin model compounds (4 -9). The oxidizing peroxide cosubstrate for this reaction must be generated in situ for efficient turnover of extracellular lignin peroxidase, a function performed by glyoxal oxidase, which catalyzes the oxidation of a number of aldehyde and ␣-hydroxy carbonyl compounds, reducing O 2 to H 2 O 2 in the process. The enzyme exhibits a broad substrate specificity for oxidation of simple aldehydes to the corresponding carboxylic acids, as shown by Reaction 1 (10).Two of the substrates for glyoxal oxidase (glyoxal (OHCCHO) and methylglyoxal (CH 3 COCHO)) are found in extracellular fluid of ligninolytic cultures (10) and are likely to represent physiological substrates for the enzyme in a complex extracellular metabolic scheme (9, 10). Purified glyoxal oxidase is catalytically inactive but can be activated by lignin peroxidase (11, 12), suggesting a possible extracellular regulatory circuit for the control of H 2 O 2 production by glyoxal oxidase, and lignin peroxidase activity by H 2 O 2 .P. chrysosporium glyoxal oxidase has been purified to homogeneity (11), cloned for high level expression in Aspergillus nidulans (13...

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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 measureme...