Acetylacetone dioxygenase from Acinetobacter johnsonii (Dke1) utilizes a non-heme Fe2+ cofactor to promote dioxygen-dependent conversion of 2,4-pentanedione (PD) into methylglyoxal and acetate. An oxidative carbon-carbon bond cleavage by Dke1 is triggered from a C-3 peroxidate intermediate that performs an intramolecular nucleophilic attack on the adjacent carbonyl group. But how does Dke1 bring about the initial reduction of dioxygen? To answer this question, we report here a reaction coordinate analysis for the part of the Dke1 catalytic cycle that involves O2 chemistry. A weak visible absorption band (epsilon approximately 0.2 mM(-1) cm(-1)) that is characteristic of an enzyme-bound Fe2+-beta-keto-enolate complex served as spectroscopic probe of substrate binding and internal catalytic steps. Transient and steady-state kinetic studies reveal that O2-dependent conversion of the chromogenic binary complex is rate-limiting for the overall reaction. Linear free-energy relationship analysis, in which apparent turnover numbers (k(app) cat) for enzymatic bond cleavage of a series of substituted beta-dicarbonyl substrates were correlated with calculated energies for the highest occupied molecular orbitals of the corresponding beta-keto-enolate structures, demonstrates unambiguously that k(app) cat is governed by the electron-donating ability of the substrate. The case of 2'-hydroxyacetophenone (2'HAP), a completely inactive beta-dicarbonyl analogue that has the enol double bond delocalized into the aromatic ring, indicates that dioxygen reduction and C-O bond formation cannot be decoupled and therefore take place in one single kinetic step.

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe(2+) is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a K (m) of 9.1 microM and a k(cat) of 8.5 s(-1). A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

A facial triad of two histidine side chains and one aspartate or glutamate side chain forms the canonical metal-coordinating motif in the catalytic centers of various mononuclear non-heme Fe(II) enzymes. Although these active sites are based on totally unrelated protein folds and bring about a wide range of chemical transformations, most of them share the ability to couple dioxygen reduction with the oxygenation of an organic substrate. With the increasing number of protein structures now solved, it has become clear that the 2-His-1-carboxylate signature is less of a paradigm for non-heme Fe(II) active sites than had long been thought and that it can be replaced by alternative metal centers in various oxygenases, the structure-function relationships and proposed catalytic mechanisms of which are reviewed here. Metal coordination through three histidines and one glutamate constitutes the classical motif described for enzyme members of the cupin protein superfamily, such as aci-reductone dioxygenase and quercetin dioxygenase, multiple metal forms of which (including the Fe(II) type) are found in nature. Cysteine dioxygenase and diketone dioxygenase, which are strictly Fe(II)-dependent oxygenases based on the cupin fold, bind the catalytic metal through the homologous triad of histidines, but lack the fourth glutamate ligand. An alpha-ketoglutarate-dependent Fe(II) halogenase shows metal coordination by two histidines as the only protein-derived ligands, whilst carotene oxygenase, from a different protein fold family, features an Fe(II) site consisting of four histidine side chains. These recently discovered metallocenters are discussed with respect to their metal-binding properties and the reaction coordinates of the O(2)-dependent conversions they catalyze.

Graphical abstractHighlights► The Cupin fold provides metal binding motif plasticity. ► The 2-His binding motif offers a platform for halogenations. ► The 3-His binding motif affects the substrate ligation mode and reactivity. ► The 3-His-1 Carboxylate Motif shows remarkable metal ion promiscuity. ► Outer shell residues impact metal center stability and catalysis.

Density functional theory calculations on the oxygen activation process in cysteine dioxygenase (CDO) and three active site mutants whereby one histidine group is replaced by a carboxylic acid group are reported. The calculations predict an oxygen activation mechanism that starts from an Fe(III)-O-O(*) complex that has close lying singlet, triplet, and quintet spin states. A subsequent spin state crossing to the quintet spin state surfaces leads to formation of a ring-structure whereby an O-S bond is formed. This weakens the central O-O bond, which is subsequently broken to give sulfoxide and an iron-oxo complex. The second oxygen atom is transferred to the substrate after a rotation of the sulfoxide group. A series of calculations were performed on cysteine dioxygenase mutants with a 2His/1Asp motif rather than a 3His motif. These calculations focused on the differences in catalytic and electronic properties of nonheme iron systems with a 3His ligand system versus a 2His/1Asp motif, such as taurine/alpha-ketoglutarate dioxygenase (TauD), and predict why CDO has a 3His ligand system while TauD and other dioxygenases share a 2His/1Asp motif. One mutant (H86D) had the ligand trans to the dioxygen group replaced by acetate, while in another set of calculations the ligand trans to the sulfur group of cysteinate was replaced by acetate (H88D). The calculations show that the ligands influence the spin state ordering of the dioxygen bound complexes considerably and in particular stabilize the quintet spin state more so that the oxygen activation step should encounter a lower energetic cost in the mutants as compared to WT. Despite this, the mutant structures require higher O-O bond breaking energies. Moreover, the mutants create more stable iron-oxo complexes than the WT, but the second oxygen atom transfer to the substrate is accomplished with much higher reaction barriers than the WT system. In particular, a ligand trans to the sulfur atom of cysteine that pushes electrons to the iron will weaken the Fe-S bond and lead to dissociation of this bond in an earlier step in the catalytic cycle than the WT structure. On the other hand, replacement of the ligand trans to the dioxygen moiety has minor effects on cysteinate binding but enhances the barriers for the second oxygen transfer process. These studies have given insight into why cysteine dioxygenase enzymes contain a 3His ligand motif rather than 2His/1Asp and show that the ligand system is essential for optimal dioxygenation activity of the substrate. In particular, CDO mutants with a 2His/1Asp motif may give sulfoxides as byproduct due to incomplete dioxygenation processes.