The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.

Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

The unique Phe-His dyad of 2-ketopropyl coenzyme M oxidoreductase/carboxylase selectively promotes carboxylation and S-C bond cleavage.

The 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) enzyme is the only member of the disulfide oxidoreductase (DSOR) family of enzymes, which are important for reductively cleaving S-S bonds, to have carboxylation activity. 2-KPCC catalyzes the conversion of 2-ketopropyl-coenzyme M to acetoacetate, which is used as a carbon source, in a controlled reaction to exclude protons. A conserved His-Glu motif present in DSORs is key in the protonation step; however, in 2-KPCC, the dyad is substituted by Phe-His. Here, we propose that this difference is important for coupling carboxylation with C-S bond cleavage. We substituted the Phe-His dyad in 2-KPCC to be more DSOR like, replacing the phenylalanine with histidine (F501H) and the histidine with glutamate (H506E), and solved crystal structures of F501H and the double variant F501H_H506E. We found that F501 protects the enolacetone intermediate from protons and that the F501H variant strongly promotes protonation. We also provided evidence for the involvement of the H506 residue in stabilizing the developing charge during the formation of acetoacetate, which acts as a product inhibitor in the WT but not the H506E variant enzymes. Finally, we determined that the F501H substitution promotes a DSOR-like charge transfer interaction with flavin adenine dinucleotide, eliminating the need for cysteine as an internal base. Taken together, these results indicate that the 2-KPCC dyad is responsible for selectively promoting carboxylation and inhibiting protonation in the formation of acetoacetate.

Substrate-Triggered Formation of a Peroxo-Fe2(III/III) Intermediate during Fatty Acid Decarboxylation by UndA.

The iron-dependent oxidase UndA cleaves one C3-H bond and the C1-C2 bond of dodecanoic acid to produce 1-undecene and CO2. A published X-ray crystal structure showed that UndA has a heme-oxygenase-like fold, thus associating it with a structural superfamily that includes known and postulated non-heme diiron proteins, but revealed only a single iron ion in the active site. Mechanisms proposed for initiation of decarboxylation by cleavage of the C3-H bond using a monoiron cofactor to activate O2 necessarily invoked unusual or potentially unfeasible steps. Here we present spectroscopic, crystallographic, and biochemical evidence that the cofactor of Pseudomonas fluorescens Pf-5 UndA is actually a diiron cluster and show that binding of the substrate triggers rapid addition of O2 to the Fe2(II/II) cofactor to produce a transient peroxo-Fe2(III/III) intermediate. The observations of a diiron cofactor and substrate-triggered formation of a peroxo-Fe2(III/III) intermediate suggest a small set of possible mechanisms for O2, C3-H and C1-C2 activation by UndA; these routes obviate the problematic steps of the earlier hypotheses that invoked a single iron.

The reactive form of a C-S bond-cleaving, CO2-fixing flavoenzyme.

NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.

Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b.

The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e-/2H+ from each propionate to an activated form of H2O2 Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme-coproheme complex with H2O2 This radical disappeared over the next 270 s, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 s after initiating the reaction with H2O2, nor did it change the ∼270 s required for the radical signal to recede to ≤10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145• was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV)=O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of ∼2 on the decarboxylation rate constant, indicating that cleavage of the propionate Cß-H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate Cß-H to Tyr-145•.

Reactions of Ferrous Coproheme Decarboxylase (HemQ) with O2 and H2O2 Yield Ferric Heme b.

A recently discovered pathway for the biosynthesis of heme b ends in an unusual reaction catalyzed by coproheme decarboxylase (HemQ), where the Fe(II)-containing coproheme acts as both substrate and cofactor. Because both O2 and H2O2 are available as cellular oxidants, pathways for the reaction involving either can be proposed. Analysis of reaction kinetics and products showed that, under aerobic conditions, the ferrous coproheme-decarboxylase complex is rapidly and selectively oxidized by O2 to the ferric state. The subsequent second-order reaction between the ferric complex and H2O2 is slow, pH-dependent, and further decelerated by D2O2 (average kinetic isotope effect of 2.2). The observation of rapid reactivity with peracetic acid suggested the possible involvement of Compound I (ferryl porphyrin cation radical), consistent with coproheme and harderoheme reduction potentials in the range of heme proteins that heterolytically cleave H2O2. Resonance Raman spectroscopy nonetheless indicated a remarkably weak Fe-His interaction; how the active site structure may support heterolytic H2O2 cleavage is therefore unclear. From a cellular perspective, the use of H2O2 as an oxidant in a catalase-positive organism is intriguing, as is the unusual generation of heme b in the Fe(III) rather than Fe(II) state as the end product of heme synthesis.

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ).

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C-C bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H2O2-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct C-H bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr•. This residue then abstracts a net hydrogen atom (H•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e-/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.

Time-resolved Studies of IsdG Protein Identify Molecular Signposts along the Non-canonical Heme Oxygenase Pathway.

IsdGs are heme monooxygenases that break open the tetrapyrrole, releasing the iron, and thereby allowing bacteria expressing this protein to use heme as a nutritional iron source. Little is currently known about the mechanism by which IsdGs degrade heme, although the products differ from those generated by canonical heme oxygenases. A synthesis of time-resolved techniques, including in proteo mass spectrometry and conventional and stopped-flow UV/visible spectroscopy, was used in conjunction with analytical methods to define the reaction steps mediated by IsdG from Staphylococcus aureus and their time scales. An apparent meso-hydroxyheme (forming with k = 0.6 min(-1), pH 7.4, 10 mm ascorbate, 10 µm IsdG-heme, 22 °C) was identified as a likely common intermediate with the canonical heme oxygenases. Unlike heme oxygenases, this intermediate does not form with added H2O2 nor does it convert to verdoheme and CO. Rather, the next observable intermediates (k = 0.16 min(-1)) were a set of formyloxobilin isomers, similar to the mycobilin products of the IsdG homolog from Mycobacterium tuberculosis (MhuD). These converted in separate fast and slow phases to ß-/δ-staphylobilin isomers and formaldehyde (CH2O). Controlled release of this unusual C1 product may support IsdG's dual role as both an oxygenase and a sensor of heme availability in S. aureus.

Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ.

A recently proposed pathway for heme b biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H2O2-dependent manner by the enzyme HemQ. Using the HemQ from Staphylococcus aureus (SaHemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H2O2 is the assumed biological oxidant, either H2O2 or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H2O2 suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out.

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications.

Rapid, high-throughput, and quantitative evaluations of biological metabolites in complex milieu are increasingly required for biochemical, toxicological, pharmacological, and environmental analyses. They are also essential for the development, testing, and improvement of new commercial chemical products. We demonstrate the application of ultra-high performance liquid chromatography-mass spectrometry (uHPLC-MS), employing an electrospray ionization source and a high accuracy quadrupole time-of-flight mass analyzer, for the identification and quantification of a series of porphyrin derivatives in liver: a matrix of particular relevance in toxicological or pharmacological testing. Exact mass is used to identify and quantify the metabolites. Chromatography enhances sensitivity and alleviates potential saturation issues by fanning out the contents of a complex sample before their injection into the spectrometer, but is not strictly necessary for the analysis. Extraction and sample treatment procedures are evaluated and matrix effects discussed. Using this method, the known mechanism of action of a well-characterized porphyrinogenic agent was verified in liver extracts from treated rats. The method was also validated for use with bacterial cells. This exact-mass method uses workhorse instruments available in many laboratories, providing a highly flexible alternative to existing HPLC- and MS/MS-based approaches for the simultaneous analysis of multiple compounds in biological media.

O(2)-evolving chlorite dismutase as a tool for studying O(2)-utilizing enzymes.

The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas's modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, in situ generation of O(2) at concentrations far exceeding 2 mM. This method was used to define the O(2) concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis, in which the enzyme's Mn(IV)/Fe(III) cofactor forms from a Mn(II)/Fe(II) complex and O(2) via a Mn(IV)/Fe(IV) intermediate, at effective O(2) concentrations as high as ~10 mM. With a more soluble receptor, myoglobin, an O(2) adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C-H-bond-cleaving Fe(IV)-oxo complex, J, in taurine:α-ketoglutarate dioxygenase and superoxo-Fe(2)(III/III) complex, G, in myo-inositol oxygenase, and the tyrosyl-radical-generating Fe(2)(III/IV) intermediate, X, in Escherichia coli RNR, were all accumulated to yields more than twice those previously attained. This means of in situ O(2) evolution permits a >5 mM "pulse" of O(2) to be generated in <1 ms at the easily accessible Cld concentration of 50 µM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O(2) addition steps.

Chlorite dismutases, DyPs, and EfeB: 3 microbial heme enzyme families comprise the CDE structural superfamily.

Heme proteins are extremely diverse, widespread, and versatile biocatalysts, sensors, and molecular transporters. The chlorite dismutase family of hemoproteins received its name due to the ability of the first-isolated members to detoxify anthropogenic ClO(2)(-), a function believed to have evolved only in the last few decades. Family members have since been found in 15 bacterial and archaeal genera, suggesting ancient roots. A structure- and sequence-based examination of the family is presented, in which key sequence and structural motifs are identified, and possible functions for family proteins are proposed. Newly identified structural homologies moreover demonstrate clear connections to two other large, ancient, and functionally mysterious protein families. We propose calling them collectively the CDE superfamily of heme proteins.

Comprehensive spectroscopic, steady state, and transient kinetic studies of a representative siderophore-associated flavin monooxygenase.

Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N(5)-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O(2). The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP(+) (t(½) = 33 min, 25 °C, pH 8). NADP(+) therefore is a likely regulator of O(2) and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N(5)-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes.

How active-site protonation state influences the reactivity and ligation of the heme in chlorite dismutase.

Chlorite dismutase catalyzes O(2) release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady-state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3-11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid-base transition was observed by UV/visible and resonance Raman (rR) spectroscopy with a pK(a) of 8.7, 2-3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five-coordinate high-spin Fe(III) to a mixed high/low-spin ferric hydroxide, as confirmed by rR spectroscopy. The two Fe-OH stretching frequencies are quite low, consistent with a weak Fe-OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe-OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond-donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pK(a) approximately 6.5) form also appears to act as the active-site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady-state profile for the rate of chlorite decomposition is characterized by these same pK(a) values. The five-coordinate high-spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum k(cat) = 2.0(+/-0.6) x 10(5) s(-1), k(cat)/K(M) = 3.2(+/-0.4) x 10(7) M(-1) s(-1), pH 5.2, 4 degrees C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions.

Structural features promoting dioxygen production by Dechloromonas aromatica chlorite dismutase.

Chlorite dismutase (Cld) is a heme enzyme capable of rapidly and selectively decomposing chlorite (ClO(2) (-)) to Cl(-) and O(2). The ability of Cld to promote O(2) formation from ClO(2) (-) is unusual. Heme enzymes generally utilize ClO(2) (-) as an oxidant for reactions such as oxygen atom transfer to, or halogenation of, a second substrate. The X-ray crystal structure of Dechloromonas aromatica Cld co-crystallized with the substrate analogue nitrite (NO(2) (-)) was determined to investigate features responsible for this novel reactivity. The enzyme active site contains a single b-type heme coordinated by a proximal histidine residue. Structural analysis identified a glutamate residue hydrogen-bonded to the heme proximal histidine that may stabilize reactive heme species. A solvent-exposed arginine residue likely gates substrate entry to a tightly confined distal pocket. On the basis of the proposed mechanism of Cld, initial reaction of ClO(2) (-) within the distal pocket generates hypochlorite (ClO(-)) and a compound I intermediate. The sterically restrictive distal pocket probably facilitates the rapid rebound of ClO(-) with compound I forming the Cl(-) and O(2) products. Common to other heme enzymes, Cld is inactivated after a finite number of turnovers, potentially via the observed formation of an off-pathway tryptophanyl radical species through electron migration to compound I. Three tryptophan residues of Cld have been identified as candidates for this off-pathway radical. Finally, a juxtaposition of hydrophobic residues between the distal pocket and the enzyme surface suggests O(2) may have a preferential direction for exiting the active site.

Crystallization and preliminary X-ray diffraction of chlorite dismutase from Dechloromonas aromatica RCB.

Chlorite dismutase from Dechloromonas aromatica RCB, a novel b-type hemoprotein that catalyzes O-O bond formation, has been crystallized. Synchrotron X-ray diffraction data have been collected to 3.0 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 122.7, b = 202.9, c = 247.1 A.

Mechanism of and exquisite selectivity for O-O bond formation by the heme-dependent chlorite dismutase.

Chlorite dismutase (Cld) is a heme b-dependent, O-O bond forming enzyme that transforms toxic chlorite (ClO(2)(-)) into innocuous chloride and molecular oxygen. The mechanism and specificity of the reaction with chlorite and alternate oxidants were investigated. Chlorite is the sole source of dioxygen as determined by oxygen-18 labeling studies. Based on ion chromatography and mass spectrometry results, Cld is highly specific for the dismutation of chlorite to chloride and dioxygen with no other side products. Cld does not use chlorite as an oxidant for oxygen atom transfer and halogenation reactions (using cosubstrates guaiacol, thioanisole, and monochlorodimedone, respectively). When peracetic acid or H(2)O(2) was used as an alternative oxidant, oxidation and oxygen atom transfer but not halogenation reactions occurred. Monitoring the reaction of Cld with peracetic acid by rapid-mixing UV-visible spectroscopy, the formation of the high valent compound I intermediate, [(Por(*+))Fe(IV) = O], was observed [k(1) = (1.28 +/- 0.04) x 10(6) M(-1) s(-1)]. Compound I readily decayed to form compound II in a manner that is independent of peracetic acid concentration (k(2) = 170 +/- 20 s(-1)). Both compound I and a compound II-associated tryptophanyl radical that resembles cytochrome c peroxidase (Ccp) compound I were observed by EPR under freeze-quench conditions. The data collectively suggest an O-O bond-forming mechanism involving generation of a compound I intermediate via oxygen atom transfer from chlorite, and subsequent recombination of the resulting hypochlorite and compound I.

Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase.

Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2- to Cl- and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (approximately 4.7 x 10(3) micromol of ClO2- min(-1) mg(-1) subunit) and nearly stoichiometric in heme; further, it shares spectroscopic and physicochemical features with chlorite dismutases previously isolated from three organisms. A careful study of the enzyme's steady state kinetics has been carried out. ClO2- consumption and O2 release rates were measured, yielding comparable values of kcat (4.5 x 10(5) min(-1)), K(m) (approximately 215 microM), and kcat/Km (3.5 x 10(7) M(-1) s(-1) via either method (4 degrees C, pH 6.8; all values referenced per heme-containing subunit). ClO2-:O2 stoichiometry exhibited a 1:1 relationship under all conditions measured. Though the value of kcat/Km indicates near diffusion control of the reaction, viscosogens had no effect on k(cat)/K(m) or V(max). The product O2 did not inhibit the reaction at saturating [O2], but Cl- is a mixed inhibitor with relatively high values of KI (225 mM for enzyme and 95.6 mM for the enzyme-substrate complex), indicating a relatively low affinity of the heme iron for halogen ions. Chlorite irreversibly inactivates the enzyme after approximately 1.7 x 10(4) turnovers (per heme) and with a half-life of 0.39 min, resulting in bleaching of the heme chromophore. The inactivation K(I) (K(inact)) of 166 microM is similar in magnitude to Km, consistent with a common Michaelis complex on the pathway to both reaction and inactivation. The one-electron peroxidase substrate guaiacol offers incomplete protection of the enzyme from inactivation. Mechanisms in keeping with the available data and the properties of other well-described heme enzymes are proposed.