4-Nitrocatechol as a probe of a Mn(II)-dependent extradiol-cleaving catechol dioxygenase (MndD): comparison with relevant Fe(II) and Mn(II) model complexes

Mn(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) is an extradiol-cleaving catechol dioxygenase from Arthrobacter globiformis that has 82% sequence identity to and cleaves the same substrate (3,4-dihydroxyphenylacetic acid) as Fe(II)-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum. We have observed that MndD binds the chromophoric 4-nitrocatechol (4-NCH(2)) substrate as a dianion and cleaves it extremely slowly, in contrast to the Fe(II)-dependent enzymes which bind 4-NCH(2) mostly as a monoanion and cleave 4-NCH(2) 4-5 orders of magnitude faster. These results suggest that the monoanionic binding state of 4-NC is essential for extradiol cleavage. In order to address the differences in 4-NCH(2) binding to these enzymes, we synthesized and characterized the first mononuclear monoanionic and dianionic Mn(II)-(4-NC) model complexes as well as their Fe(II)-(4-NC) analogs. The structures of [(6-Me(2)-bpmcn)Fe(II)(4-NCH)](+), [(6-Me(3)-TPA)Mn(II)(DBCH)](+), and [(6-Me(2)-bpmcn)Mn(II)(4-NCH)](+) reveal that the monoanionic catecholate is bound in an asymmetric fashion (Delta r(metal-O(catecholate))=0.25-0.35 A), as found in the crystal structures of the E(.)S complexes of extradiol-cleaving catechol dioxygenases. Acid-base titrations of [(L)M(II)(4-NCH)](+) complexes in aprotic solvents show that the p K(a) of the second catecholate proton of 4-NCH bound to the metal center is half a p K(a) unit higher for the Mn(II) complexes than for the Fe(II) complexes. These results are in line with the Lewis acidities of the two divalent metal ions but are the opposite of the trend observed for 4-NCH(2) binding to the Mn(II)- and Fe(II)-catechol dioxygenases. These results suggest that the MndD active site decreases the second p K(a) of the bound 4-NCH(2) relative to the HPCD active site.     

In vivo self-hydroxylation of an iron-substituted manganese-dependent extradiol cleaving catechol dioxygenase

The homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (MndD) catalyzes the oxidative ring cleavage reaction of its catechol substrate in an extradiol fashion. Although this reactivity is more typically associated with non-heme iron enzymes, MndD exhibits an unusual specificity for manganese(II). MndD is structurally very similar to the iron(II)-dependent homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD), and we have previously shown that both MndD and HPCD are equally active towards substrate turnover with either iron(II) or manganese(II) (Emerson et al. in Proc. Natl. Acad. Sci. USA 105:7347–7352, 2008). However, expression of MndD in Escherichia coli under aerobic conditions in the presence of excess iron results in the isolation of inactive blue-green iron-substituted MndD. Spectroscopic studies indicate that this form of iron-substituted MndD contains an iron(III) center with a bound catecholate, which is presumably generated by in vivo self-hydroxylation of a second-sphere tyrosine residue, as found for other self-hydroxylated non-heme iron oxygenases. The absence of this modification in either the native manganese-containing MndD or iron-containing HPCD suggests that the metal center of iron-substituted MndD is able to bind and activate O₂ in the absence of its substrate, employing a high-valence oxoiron oxidant to carry out the observed self-hydroxylation chemistry. These results demonstrate that the active site metal in MndD can support two dramatically different O₂ activation pathways, further highlighting the catalytic flexibility of enzymes containing a 2-His-1-carboxylate facial triad metal binding motif.     

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.     

Theoretical study of the catalytic reaction mechanism of MndD

Manganese-dependent homoprotocatechuate 2,3-dioxygenase (MndD) is an enzyme taking part in the catabolism of aromatic compounds in the environment. It uses molecular oxygen to perform an extradiol cleavage of the ring of the ortho-dihydroxylated aromatic compound homoprotocatechuate. A theoretical investigation of the reaction path for MndD was performed using hybrid density functional theory with the B3LYP functional, and a catalytic mechanism has been suggested. Models of different size were built from the crystal structure of the enzyme and were used in the search for intermediates and transition states. It was found that the substrate first binds at the active site as a monoanion. Next the dioxygen is bound, forming a hydroperoxo intermediate. The O–O bond, activated in this way undergoes homolytic cleavage leading to an oxyl and then to an extra epoxide radical with subsequent opening of the aromatic ring. The lactone ring is then hydrolyzed by the Mn-bound OH group, and the final product is obtained in the last reaction steps. Alternative reaction paths were considered, and their calculated barriers were found to be higher than for the suggested mechanism. The selectivity between the extra- and intra-cleavage pathways was found to be determined by the barriers for the decay of the radical state.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

On the oxidation of 3,4-dihydroxyphenethyl alcohol and 3,4-dihydroxyphenyl glycol by cuticular enzyme(s) from Sarcophaga bullata

The catabolic fate of 3,4-dihydroxyphenethyl alcohol (DHPA) and 3,4-dihydroxyphenylethyl glycol (DHPG) in insect cuticle was determined for the first time using cuticular enzyme(s) from Sarcophaga bullata and compared with mushroom tyrosinase-medicated oxidation. Mushroom tyrosinase converted both DHPA and DHPG to their corresponding quinone derivatives, while cuticular enzyme(s) partly converted DHPA to DHPG. Cuticular enzyme(s)-mediated oxidation of DHPA also accompanied the covalent binding of DHPA to the cuticle. Cuticle-DHPA adducts, upon pronase digestion, released peptides that had bound catechols. 3,4-Dihydroxyphenyl-acetaldehyde, the expected product of side chain desaturation of DHPA, was not formed at all. The presence of N-acetylcysteine, a quinone trap, in the reaction mixture containing DHPA and cuticle resulted in the generation of DHPA-quinone-N-acetylcysteine adduct and total inhibition of DHPG formation. The insect enzyme(s) converted DHPG to its quinone at high substrate concentration and to 2-hydroxy-3′,4′-dihydroxyacetophenone at low concentration. They converted exogenously added DHPA-quinone to DHPG, but acted sluggishly on DHPG-quinone. These results are consistent with the enzymatic transformations of phenoloxidase-generated quinones to quinone methides and subsequent nonenzymatic transformation of the latter to the observed products. Thus, quinone methide formation in insect cuticle seems to be caused by the combined action of two enzymes, phenoloxidase and quinone tautomerase, rather than the action of quinone methide-generating phenoloxidase (Sugumaran: Arch Insect Biochem Physiol 8, 73–88, 1988). It is proposed that DHPA and DHPG in combination can be used effectively to examine the participation of (1) quinone, (2) quinone methide, and (3) dehydro derivative intermediates in the metabolism of 4-alkylcatechols for cuticular sclerotization.     

Brown pigmentation in Serratia marcescens cultures associated with tyrosine metabolism

Serratia marcescens produced a brown pigment when grown in minimal medium in the presence of tyrosine and high concentrations of copper(II) ion. The pigment was not related to the melanin pigments, but was similar to the pigment produced by autooxidation and polymerization of 3,4-dihydroxyphenylacetate, which is synthesized in S. marcescens from tyrosine through the 3,4-dihydroxyphenylacetate catabolic pathway. The enzymes of this pathway were induced under pigment production conditions; however, 3,4-dihydroxyphenylacetate 2,3-dioxygenase remained at low activity levels, permitting the accumulation and excretion of the substrate. Mutants unable to use tyrosine as a sole carbon and energy source were able to produce brown pigments only if the step blocked by the mutation was after the synthesis of 3,4-dihydroxyphenylacetate. The ability to produce brown pigments was common to all the S. marcescens strains tested.     

NMR study of manganese(II) binding by a new versatile peroxidase from the white-rot fungus<Emphasis Type="Italic"> Pleurotus eryngii</Emphasis>

Nuclear magnetic resonance spectroscopy has been used to characterize the versatile peroxidase from Pleurotus eryngii, both in the resting state and in the cyanide-inhibited form. The assignment of most of the hyperfine-shifted resonances has been achieved by two-dimensional NMR, allowing the comparison of the present system with other ligninolytic peroxidases. This information has enabled a detailed analysis of the interaction of the enzyme with one of its reducing substrates, Mn(II). Furthermore, comparison with the data collected on a mutant in the putative Mn(II) binding site, and an analysis of the enzyme kinetic properties, shed light on the factors affecting the function of this novel peroxidase.Electronic Supplementary Material Supplementary material is available for this article if you access the article at .Abbreviations ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonate) - CcP cytochrome c peroxidase - CIP Coprinus cinereus peroxidase - HRP horseradish peroxidase - IPTG isopropyl--D-thiogalactopyranoside - LiP lignin peroxidase - MnP manganese peroxidase - RB5 Reactive Black 5 - VA veratryl alcohol (3,4-dimethoxybenzyl alcohol) - VP versatile peroxidase     

Biochemical identification of residues that discriminate between 3,4-dihydroxyphenylalanine decarboxylase and 3,4-dihydroxyphenylacetaldehyde synthase-mediated reactions

In available insect genomes, there are several L-3,4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC)-like or aromatic amino acid decarboxylase (AAAD) sequences. This contrasts to those of mammals whose genomes contain only one DDC. Our previous experiments established that two DDC-like proteins from Drosophila actually mediate a complicated decarboxylation-oxidative deamination process of dopa in the presence of oxygen, leading to the formation of 3,4-dihydroxyphenylacetaldehyde (DHPA), CO₂, NH_3, and H₂O₂. This contrasts to the typical DDC-catalyzed reaction, which produces CO₂ and dopamine. These DDC-like proteins were arbitrarily named DHPA synthases based on their critical role in insect soft cuticle formation. Establishment of reactions catalyzed by these AAAD-like proteins solved a puzzle that perplexed researchers for years, but to tell a true DHPA synthase from a DDC in the insect AAAD family remains problematic due to high sequence similarity. In this study, we performed extensive structural and biochemical comparisons between DHPA synthase and DDC. These comparisons identified several target residues potentially dictating DDC-catalyzed and DHPA synthase-catalyzed reactions, respectively. Comparison of DHPA synthase homology models with crystal structures of typical DDC proteins, particularly residues in the active sites, provided further insights for the roles these identified target residues play. Subsequent site-directed mutagenesis of the tentative target residues and activity evaluations of their corresponding mutants determined that active site His192 and Asn192 are essential signature residues for DDC- and DHPA synthase-catalyzed reactions, respectively. Oxygen is required in DHPA synthase-mediated process and this oxidizing agent is reduced to H₂O₂ in the process. Biochemical assessment established that H₂O₂, formed in DHPA synthase-mediated process, can be reused as oxidizing agent and this active oxygen species is reduced to H₂O; thereby avoiding oxidative stress by H₂O₂. Results of our structural and functional analyses provide a reasonable explanation of mechanisms involved in DHPA synthase-mediated reactions. Based on the key active site residue Asn192, identified in Drosophila DHPA synthase, we were able to distinguish all available insect DHPA synthases from DDC sequences primarily.     

Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities

Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).     

Degradation of (+/-)-synephrine by Arthrobacter synephrinum. Oxidation of 3,4-dihydroxyphenylacetate to 2-hydroxy-5-carboxymethyl-muconate semialdehyde.

1. Cell-free extracts of Arthrobacter synephrinum catalyse the oxidation of 3,4-dihydroxy-phenylacetate. 2. The product of oxidation was characterized as 2-hydroxy-5-carboxymethylmuconate semialdehyde from its chemical behaviour as well as from nuclear-magnetic-resonance spectra. 3. A 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15) was partially purified from A. synephrinum. 4. The enzyme had a Km of 25 micrometer towards its substrate and exhibited typical Michaelis-Menten kinetics. 5. The enzyme also catalysed the oxidation of 3,4-dihydroxymandelate and 3,4-dihydroxyphenylpropionate, at reaction rates of 0.5 and 0.04 respectively of that for 3,4-dihydroxyphenylacetate. 6. The enzyme was sensitive to treatment with thiol-specific reagents. 7. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography was approx. 282000.     

Catecholase activity of a μ-hydroxodicopper(II) macrocyclic complex: structures, intermediates and reaction mechanism

The monohydroxo-bridged dicopper(II) complex (1), its reduced dicopper(I) analogue (2) and the trans-μ-1,2-peroxo-dicopper(II) adduct (3) with the macrocyclic N-donor ligand [22]py4pz (9,22-bis(pyridin-2′-ylmethyl)-1,4,9,14,17,22,27,28,29,30- decaazapentacyclo -[22.2.1^14,7.1^11,14.1^17,20]triacontane-5,7(28),11(29),12,18,20(30), 24(27),25-octaene), have been prepared and characterized, including a 3D structure of 1 and 2. These compounds represent models of the three states of the catechol oxidase active site: met, deoxy (reduced) and oxy. The dicopper(II) complex 1 catalyzes the oxidation of catechol model substrates in aerobic conditions, while in the absence of dioxygen a stoichiometric oxidation takes place, leading to the formation of quinone and the respective dicopper(I) complex. The catalytic reaction follows a Michaelis–Menten behavior. The dicopper(I) complex binds molecular dioxygen at low temperature, forming a trans-μ-1,2-peroxo-dicopper adduct, which was characterized by UV–Vis and resonance Raman spectroscopy and electrochemically. This peroxo complex stoichiometrically oxidizes a second molecule of catechol in the absence of dioxygen. A catalytic mechanism of catechol oxidation by 1 has been proposed, and its relevance to the mechanisms earlier proposed for the natural enzyme and other copper complexes is discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Synthesis, structure and interactions with DNA of novel tetranuclear, [Mn4(II/II/II/IV)] mixed valence complexes

Reaction of Mn(II) with phenoxyalkanoic acids and di-2-pyridyl ketone oxime (Hpko) leads to neutral tetranuclear complexes of the general formula Mn(4)(O)(pko)(4)(phenoxyalkanoato)(4) (phenoxyalkanoic acids: H-mcpa=2-methyl-4-chloro-phenoxy-acetic acid, H-2,4,5-T=2,4,5-trichloro-phenoxy-acetic acid or H3,4-D=3,4-dichloro-phenoxy-acetic acid). The compounds were synthesized by adding di-2-pyridyl ketone oxime to MnCl(2) in the presence of the sodium salts of the alkanoic acids in methanol. The crystal structure of Mn(4)(II/II/II/IV)(O)(pko)(4)(2,4,5-T)(4).2.5CH(3)OH.0.25H(2)O 1 shows that the complex consists of a [Mn(4)(mu(4)-O)](8+) core with a Mn(IV) and 3 Mn(II) ions in octahedral environment and a mu(4)-O atom bridging the four manganese ions. Spectroscopic studies of the interaction of these tetranuclear clusters with DNA showed that these compounds bind to dsDNA. The binding strength of the Mn(4)(II/II/II/IV)(O)(pko)(4)(2,4,5-T)(4) complex for calf thymus DNA is equal to 1.1x10(4)M(-1). Among the deoxyribonucleotides they bind preferentially to deoxyguanylic acid (dGMP). Competitive studies with ethidium bromide (EthBr) showed that the Mn(4)(II/II/II/IV)(O)(pko)(4)(2,4,5-T)(4) complex exhibited the ability to displace the DNA-bound EthBr indicating that the complex binds to DNA via intercalation in strong competition with EthBr for the intercalative binding site. Additionally, DNA electrophoretic mobility experiments showed that all three complexes, at low cluster concentration, are obviously capable of binding to pDNA causing its cleavage (relaxation) at physiological pH and temperature. At higher cluster concentration, catenated dimer forms of pDNA was formed.     

Improving Dioxygenase Stability by Gene Chromosome Insertion: Implementation in Immobilized-Cell Systems

The immobilization of recombinant cells by using the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase was studied as a model. Dioxygenase activity and cell viability were compared in immobilized-cell systems and cells in suspension. Immobilization increased enzyme stability and the efficient degradation of 3,4-dihydroxyphenylacetate. The stability of the cloned enzyme and the viability of the immobilized recombinant cells were well maintained for at least 15 days. We used the strain Escherichia coli CC118-D in which the hpaB gene from Klebsiella pneumoniae, coding for the subunit of 3,4-dihydroxyphenylacetate 2,3-dioxygenase, was inserted into the chromosome. This study has demonstrated that the implementation of E. coli CC118-D in a pilot-scale bioreactor resulted in a 100% stabilization of dioxygenase activity, and could be a useful tool for bioremediation processes.     

Isolation from urine of two Serratia marcescens strains excreting a diffusible yellow pigment

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.     

The effect of some 9-substituted adenine derivatives on the development of seedling roots of broad bean

Seedlings ofVicia faba L. were cultured on diluted Knop’s solution containing one of the tested substances: 9-(2,3-dihydroxypropyl)adenine (DHPA), 3-0-phosphonylmethyl DHPA (DHPA phosphonate), 3-(adenin-9-yl)-2-hydroxypropanoic acid and its esters, (2R,3R)-4-(adenin-9-yl)-2,3-dihydroxybutanoic acid (D-eritadenine) and its methyl ester. The development of main and side roots was checked. Most of the substances tested were more powerful inhibitors than DHPA. The results are discussed in connection with the role of S-adenosyl-L-methionine in plant objects.     

The Cys-Xaa-His metal-binding motif: {<Emphasis Type="Italic">N</Emphasis>} versus {<Emphasis Type="Italic">S</Emphasis>} coordination and nickel-mediated formation of cysteinyl sulfinic acid

A series of peptide ligands containing the sequence -Cys-Xaa-His- (CXH; Xaa=Gly or Lys) has been prepared and the coordination chemistry of these peptides with nickel(II) investigated. Selective protection of either the N-terminal cysteine thiol or amine group gave complexes with amino or thiolato coordination, respectively, to nickel(II). Insertion of CGH into a pentapeptide, N-acetyl-Ala-Cys-Gly-His-Ala-CONH₂, allowed the formation of a square-planar thiolato Cys-Gly-His complex with nickel(II) in an internal position of the peptide. Inclusion of an N-terminal cysteine residue with a free amino terminus gave rise to pH- and dioxygen-dependent coordination behavior. Solutions of CGH-CONH₂ with nickel(II) at neutral pH yielded a red nickel-thiolate complex, but at higher pH (8.5 or above) or with exposure to dioxygen, yellow nickel complexes with N-terminal amino coordination were observed. The disulfide-bridged dimers formed from Ni(CGH-CONH₂) in the presence of air were characterized and found to have the typical coordination found in the amino-terminal binding motif of the serum albumins. Nickel(II) coordination and thiol reactivity were also studied by determination of rates of thiol alkylation and by monitoring air oxidation in the presence of various metals. Zinc(II) effectively inhibits thiol alkylation and oxidation (disulfide formation) in all the peptides studied. Nickel(II) inhibits aerobic oxidation and alkylation of N-terminal protected peptides such as N-acetyl-Cys-Gly-His, but does not inhibit air oxidation of free amino terminal peptides such as Cys-Gly-His. Instead, nickel(II) mediates the formation an additional product under aerobic conditions, a cysteinesulfinic acid.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations CGH cysteinylglycylhistidine - GGH glycylglycylhistidine - Xaa any amino acid     

Water, water everywhere, and its remarkable chemistry

Photosystem II (PSII), the multisubunit pigment-protein complex localised in the thylakoid membranes of oxygenic photosynthetic organisms, uses light energy to drive a series of remarkable reactions leading to the oxidation of water. The products of this oxidation are dioxygen, which is released to the atmosphere, and reducing equivalents destined to reduce carbon dioxide to organic molecules. The water oxidation occurs at catalytic sites composed of four manganese atoms (Mn(4)-cluster) and powered by the redox potential of an oxidised chlorophyll a molecule (P680(*+)). Gerald T (Jerry) Babcock and colleagues showed that electron/proton transfer processes from substrate water to P680(*+) involved a tyrosine residue (Y(Z)) and proposed an attractive reaction mechanism for the direct involvement of Y(Z) in the chemistry of water oxidation. The 'hydrogen-atom abstract/metalloradical' mechanism he formulated is an expression of his genius and a highlight of his many other outstanding contributions to photosynthesis research. A structural basis for Jerry's model is now being revealed by X-ray crystallography.     

Five-coordinated metal complexes of bis(2-hydroxy-1-naphthylideneimine-3-propyl)amine and their reactivity towards dioxygen. Part I. An electrochemical investigation on manganese(II), iron(II), cobalt(II), nickel(II) and copper(II) complexes

The electrochemical behaviour in aprotic solvent of the complexes {M[bis-(2-hydroxy-l-naphthylideneimine-3-propyl)amine]}, where M = Mn(II), Co(II), Fe(II), Ni(II) and Cu(II) is reported. The complexes were prepared and characterized by elemental analysis, infrared and visible spectroscopy and magnetic susceptibility measurements. In addition the reactivity towards dioxygen of the Mn(II), Fe(II) and Co(II) derivatives was investigated, mainly by cyclic voltammetry and gas-volumetric uptake measurements. The results indicate that the Co(II) complexes are able to add dioxygen reversibly, while Mn(II) and Fe(II) compounds undergo an irreversible oxygenation process. The pathway of the dioxygenation processes is tentatively interpreted on the basis of the electrochemical responses. The results confirm that the location of the oxidation potential allows one to predict whether a compound is able to react with dioxygen, but it is not sufficient to predict whether the dioxygenation reaction proceeds reversibly.     

Catecholase activity of dicopper(II)-bispidine complexes: stabilities and structures of intermediates,kinetics and reaction mechanism

A mechanism for the oxidation of 3,5-di-tert-butylcatechol (dtbc) with dioxygen to the corresponding quinone (dtbq), catalyzed by bispidine-dicopper complexes (bispidines are various mono- and dinucleating derivatives of 3,7-diazabicyclo[3.3.1]nonane with bis-tertiary-amine–bispyridyl or bis-tertiary-amine–trispyridyl donor sets), is proposed on the basis of (1) the stoichiometry of the reaction as well as the stabilities and structures [X-ray, density functional theory (B3LYP, TZV)] of the bispidine-dicopper(II)–3,4,5,6-tetrachlorcatechol intermediates, (2) formation kinetics and structures (molecular mechanics, MOMEC) of the end-on peroxo–dicopper(II) complexes and (3) kinetics of the stoichiometric (anaerobic) and catalytic (aerobic) copper-complex-assisted oxidation of dtbc. This involves (1) the oxidation of the dicopper(I) complexes with dioxygen to the corresponding end-on peroxo–dicopper(II) complexes, (2) coordination of dtbc as a bridging ligand upon liberation of H₂O₂ and (3) intramolecular electron transfer to produce dtbq, which is liberated, and the dicopper(I) catalyst. Although the bispidine complexes have reactivities comparable to those of recently published catalysts with macrocyclic ligands, which seem to reproduce the enzyme-catalyzed process in various reaction sequences, a strikingly different oxidation mechanism is derived from the bispidine–dicopper-catalyzed reaction. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfocatechol

4-Aminobenzenesulfonate is degraded via 4-sulfocatechol by a mixed bacterial culture that consists of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. From the 4-sulfocatechol-degrading organism A. radiobacter strain S2, a dioxygenase that converted 4-sulfocatechol to 3-sulfomuconate was purified to homogeneity. The purified enzyme also converted protocatechuate with a similar catalytic activity to 3-carboxy-cis,cis-muconate. Furthermore, the purified enzyme oxidized 3,4-dihydroxyphenylacetate, 3,4-dihydroxycinnamate, catechol, and 3- and 4-methylcatechol. The enzyme had a mol. wt. of about 97,400 as determined by gel filtration and consisted of two different types of subunits with mol. wt. of about 23,000 and 28,500. The NH₂-terminal amino acid sequences of the two subunits were determined. An isofunctional dioxygenase was partially purified from H. palleronii strain S1. A. radiobacter strain S2 also induced, after growth with 4-sulfocatechol, an „ordinary“ protocatechuate 3,4-dioxygenase that did not oxidize 4-sulfocatechol. This enzyme was also purified to homogeneity, and its catalytic and structural characteristics were compared to the „4-sulfocatechol-dioxygenase“ from the same strain. Received: 5 February 1996 / Accepted: 18 April 1996