The dynamic role of distal side residues in heme hydroperoxidase catalysis. Interplay between X-ray crystallography and ab initio MD simulations

The enzymatic cycle of hydroperoxidases involves the resting Fe(III) state of the enzyme and the high-valent iron intermediates Compound I and Compound II. These states might be characterized by X-ray crystallography and the transition pathways between each state can be investigated using atomistic simulations. Here we review our recent work in the modeling of two key steps of the enzymatic reaction of hydroperoxidases: the formation of Cpd I in peroxidase and the reduction of Cpd I in catalase. It will be shown that small conformational motions of distal side residues (His in peroxidases and His/Asn in catalases), not,or only partially, revealed by the available X-ray structures, play an important role in the catalytic processes examined.     

Replacement of tyrosine residues by phenylalanine in cytochrome P450cam alters the formation of Cpd II-like species in reactions with artificial oxidants

Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [Spolitak et al. (2005) J Biol Chem 280:20300-20309; (2006) J Inorg Biochem 100:2034-2044] spectrally characterized compound I [ferryl iron plus a porphyrin pi-cation radical (Fe(IV) = O/Por(+))], as well as Cpd ES (Fe(IV) = O/Tyr.). In the present studies, we report how the substitutions in Y75F, Y96F, and Y96F/Y75F P450cam variants permit the formation of a species we attribute to Cpd II (Fe(IV) = O) in reactions with peracids and cumene hydroperoxide. These variants produce changes in hydrogen bonding patterns and increased hydrophobicity that affect the ratio of heterolytic to homolytic pathways in reactions with cumene hydroperoxide, resulting in a shift of this ratio from 84/16 for WT to 72/28 for the Y96F/Y75F double mutant. Various ways of generating the Cpd II-like species were explored, and it was possible, especially with the more hydrophobic variants, to generate large fractions of the P450cam variants as Cpd II. The Cpd II-like species is ineffective at hydroxylating camphor, but can be readily reduced by ascorbate (as well as other peroxidase substrates) to ferric P450cam, which could then bind camphor to form the high-spin heme. The difference in the spectral properties of Cpd ES and Cpd II was rationalized as possibly being due to different states of protonation.     

Horse heart myoglobin catalyzes the H2O2-dependent oxidative dehalogenation of chlorophenols to DNA-binding radicals and quinones

The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated. Specifically, 2,4,6-trichlorophenol (TCP) is converted to 2,6-dichloro-1,4-benzoquinone in a H2O2-dependent process. The fact that Mb Cpd II is an active oxidant in halophenol dehalogenation is consistent with a traditional peroxidase order of addition of H2O2 followed by TCP. With 4-chlorophenol, a dimerized product is formed, consistent with a mechanism involving generation of a reactive phenoxy radical intermediate by an electron transfer process. The radical nature of this process may be physiologically relevant since recent studies have revealed that phenoxy radicals and electrophilic quinones, specifically of the type described herein, covalently bind to DNA [Dai, J., Sloat, A. L., Wright, M. W., and Manderville, R. A. (2005) Chem. Res. Toxicol. 18, 771-779]. Thus, the stability of Mb Cpd II and its ability to oxidize TCP may explain why such compounds are carcinogenic. Furthermore, the initial rate of dehalogenation catalyzed by Mb Cpd II is nearly comparable to that of the same reaction carried out by turnover of the ferric state, demonstrating the potential physiological danger of this long-lived, high-valent intermediate.     

Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique.

Copper-induced oxidative damage is generally attributed to the formation of the highly reactive hydroxyl radical by a mechanism analogous to the Haber-Weiss cycle for Fe(II) and H2O2. In the present work, the reaction between the Cu(I) ion and H2O2 is studied using the EPR spin-trapping technique. The hydroxyl radical adduct was observed when Cu(I), dissolved in acetonitrile under N2, was added to pH 7.4 phosphate buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Formation of the hydroxyl radical was dependent on the presence of O2 and subsequent formation of H2O2. The kscav/kDMPO ratios obtained were below those expected for a mechanism involving free hydroxyl radical and reflect the interference of nucleophilic addition of H2O to DMPO to form the DMPO/.OH adduct in the presence of nonchelated copper ion. Addition of ethanol or dimethyl sulfoxide to the reaction suggests that a high-valent metal intermediate, possibly Cu(III), was also formed. Spin trapping of hydroxyl radical was almost completely inhibited upon addition of Cu(I) to a solution of either nitrilotriacetate or histidine, even though the copper was fully oxidized to Cu(II) and H2O2 was formed. Bathocuproinedisulfonate, thiourea, and reduced glutathione all stabilized the Cu(I) ion toward oxidation by O2. Upon addition of H2O2, the Cu(I) in all three complexes was oxidized to varying degrees; however, only the thiourea complex was fully oxidized within 2 min of reaction and produced detectable hydroxyl radicals. No radicals were detected from the bathocuproinedisulfonate or glutathione complexes. Overall, these results suggest that the deleterious effects of copper ions in vivo are diminished by biochemical chelators, especially glutathione, which probably has a major role in moderating the toxicological effects of copper.     

Microperoxidase 8 catalyzed nitration of phenol by nitrogen dioxide radicals.

Microperoxidase 8 (MP8) is a heme octapeptide obtained by hydrolytic digestion of horse heart cytochrome c. At pH below 9, the heme iron is axially coordinated to the imidazole side chain of His18 and to a water molecule. Replacement of this weak ligand by H2O2 allows the formation of high-valent iron-oxo species which are responsible for both peroxidase-like and cytochrome P450-like activities of MP8. This paper shows that MP8 is able to catalyze the nitration of phenol by nitrite. The reaction requires H2O2 and is inhibited by ligands having a high affinity for the iron, catalase and radical scavengers. This suggests that the nitrating species could be NO2* radicals formed by the oxidation of nitrite by high-valent iron-oxo species. This new activity of MP8 opens a new access to nitro-aromatic compounds under mild conditions and validates the use of this minienzyme to mimick heme peroxidases, especially in the reactions of NO-derived species with biomolecules under oxidative stress conditions.     

Functional importance of tyrosine 294 and the catalytic selectivity for the bis-Fe(IV) state of MauG revealed by replacement of this axial heme ligand with histidine

The diheme enzyme MauG catalyzes the posttranslational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. It catalyzes three sequential two-electron oxidation reactions which proceed through a high-valent bis-Fe(IV) redox state. Tyr294, the unusual distal axial ligand of one c-type heme, was mutated to His, and the crystal structure of Y294H MauG in complex with preMADH reveals that this heme now has His-His axial ligation. Y294H MauG is able to interact with preMADH and participate in interprotein electron transfer, but it is unable to catalyze the TTQ biosynthesis reactions that require the bis-Fe(IV) state. This mutation affects not only the redox properties of the six-coordinate heme but also the redox and CO-binding properties of the five-coordinate heme, despite the 21 ? separation of the heme iron centers. This highlights the communication between the hemes which in wild-type MauG behave as a single diheme unit. Spectroscopic data suggest that Y294H MauG can stabilize a high-valent redox state equivalent to Fe(V), but it appears to be an Fe(IV)═O/π radical at the five-coordinate heme rather than the bis-Fe(IV) state. This compound I-like intermediate does not catalyze TTQ biosynthesis, demonstrating that the bis-Fe(IV) state, which is stabilized by Tyr294, is specifically required for this reaction. The TTQ biosynthetic reactions catalyzed by wild-type MauG do not occur via direct contact with the Fe(IV)═O heme but via long-range electron transfer through the six-coordinate heme. Thus, a critical feature of the bis-Fe(IV) species may be that it shortens the electron transfer distance from preMADH to a high-valent heme iron.     

First-principles molecular dynamics study on the atomistic behavior of His503 in bovine cytochrome c oxidase

We report first-principles molecular dynamics calculations based on density functional theory performed on the entrance part of the D-path pathway in bovine cytochrome c oxidase. Our models, which are extracted from the fully reduced and oxidized X-ray structures, include His503 as a protonatable site. We find that the protonated His503 with the deprotonated Asp91 [H503-N(δ1)H(+) and D91-C(γ)OO(γ)] are more energetically favorable than other protonation states, [H503-N(δ1) and D91-C(γ)OOH], with an energy difference of about -5kcal/mol in reduced case, while the [H503-N(δ1)H+ and D91-C(γ)OO(-)] state is energetically unstable, about +3kcal/mol higher in energy in the oxidized case. The local interaction of His503 with the surrounding polar residues is necessary and sufficient for determining the energetics. The redox-coupled rotation of His503 is found to change the energetics of the protonation states. We also find that this rotation is coupled with the proton transfer from His503 and Asp91, which leads to the transition between the two different protonation states. This study suggests that His503 is involved in the proton supply to the D-path as a proton acceptor and that the redox-controlled proton-transfer-coupled rotation of His503 is a key process for an effective proton supply to the D-path from water bulk. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

A mechanism for oxygen exchange between ligated oxometalloporphinates and bulk water

Oxygen exchange between high-valent metal-oxo complexes and bulk water has been monitored for nonligated model porphyrins (hemin, FeTDCPPS, MnTMPyP) and the axially ligated microperoxidase-8 (MP-8). Exchange extents up to 90% were measured for MP-8 in spite of the presence of an axial histidine ligand and accompanied by the formation of nonlabelled H(2)O(2) from H(2)(18)O(2). These results point to the existence of a mechanism for oxygen exchange between the high-valent iron-oxo complex and the solvent different from the so-called "oxo-hydroxo tautomerism." Regeneration of the primary oxidant, H(2)O(2), and oxygen exchange by axially ligated porphyrins can be explained by a mechanism involving the reversibility of compound I formation.     

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.     

Reaction of ferric cytochrome P450cam with peracids: kinetic characterization of intermediates on the reaction pathway

Reactions of substrate-free ferric cytochrome P450cam with peracids to generate Fe=O intermediates have previously been investigated with contradictory results. Using stopped-flow spectrophotometry, the reaction with m-chloroperoxybenzoic acid demonstrated an Fe(IV)=O + porphyrin pi-cation radical (Cpd I) (Egawa, T., Shimada, H., and Ishimura, Y. (1994) Biochem. Biophys. Res. Commun. 201, 1464-1469). By contrast, with peracetic acid, Fe(IV)=O plus a tyrosyl radical were observed by freeze-quench Mossbauer and EPR spectroscopy (Schunemann, V., Jung, C., Trautwein, A. X., Mandon, D., and Weiss, R. (2000) FEBS Lett. 479, 149-154). Our detailed kinetic studies have resolved these contradictory results. At pH >7, a significant fraction of Cpd I is formed transiently, whereas at low pH only a species with a Soret band at 406 nm, presumably Fe(IV)=O + tyrosyl radical, is observed. Evidence for formation of an acylperoxo complex en route to Cpd I was obtained. Because of rapid heme destruction, steps subsequent to formation of the highly oxidized forms could not be fully characterized. Heme destruction was avoided by including peroxidase substrates (e.g. guaiacol), which were oxidized to characteristic peroxidase products as the Fe(III)-P450 was regenerated. Addition of ascorbate to either of the high valent species also reforms the Fe(III) state with only a small loss of heme absorbance. These results indicate that typical peroxidase chemistry occurs with P450cam and offer an explanation for the contrasting results reported earlier. The delineation of improved conditions (pH, temperature, choice of peracid) for generating highly oxidized species with P450cam should be valuable for their further characterization.     

Evidence for nonhydrogen bonded compound II in cyclic reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

Studies that elucidate the behavior of the hemoglobins (Hbs) and myoglobins upon reaction with hydrogen peroxide are essential to the development of oxygen carrier substitutes. Stopped-flow kinetics and resonance Raman data show that the reaction between hydrogen peroxide and oxygenated and deoxygenated ferric Hb I (oxy- and deoxy-HbI) from Lucina pectinata produce compound I and compound II ferryl species. The rate constants ratio (k23/k41) between the formation of compound II from compound I (k23) and the oxidation of the ferrous HbI (k41, i.e., 25 M(-1) s(-1)) of 12 x 10(-4) M suggests that HbI has a peroxidative capacity for removing H2O2 from solution. Resonance Raman presents the formation of both, met-aquo-HbI and compound II ferryl species in the cyclic reaction of HbI with H2O2. The ferric HbI species is maintained by the presence of H2O2; it can produce HbI compound I, or it can be reduced to a deoxy-HbI derivative to form HbI compound II upon reaction with H2O2. The compound II ferryl vibration frequency appears at 805 and 769 cm(-1) for HbIFe(IV)=(16)O and HbIFe(IV)=(18)O species, respectively. This ferryl mode indicates the absence of hydrogen bonding between the carbonyl group of the distal Q64 and the HbIFe(IV)=O ferryl moiety. The observation suggests that both the trans-ligand effect and the polarizabilty of the HbI heme pocket are responsible for the observed ferryl oxo vibrational energy. The vibrational mode also suggests that the carbonyl group of the distal Q64 is oriented toward the iron of the heme group, increasing the distal pocket electron density.     

Crystal structures of ferredoxin variants exhibiting large changes in [Fe-S] reduction potential

Elucidating how proteins control the reduction potentials (E0') of [Fe--S] clusters is a longstanding fundamental problem in bioinorganic chemistry. Two site-directed variants of Azotobacter vinelandii ferredoxin I (FdI) that show large shifts in [Fe--S] cluster E0' (100--200 mV versus standard hydrogen electrode (SHE)) have been characterized. High resolution X-ray structures of F2H and F25H variants in their oxidized forms, and circular dichroism (CD) and electron paramagnetic resonance (EPR) of the reduced forms indicate that the overall structure is not affected by the mutations and reveal that there is no increase in solvent accessibility nor any reorientation of backbone amide dipoles or NH--S bonds. The structures, combined with detailed investigation of the variation of E0' with pH and temperature, show that the largest increases in E0' result from the introduction of positive charge due to protonation of the introduced His residues. The smaller (50--100 mV) increases observed for the neutral form are proposed to occur by directing a Hdelta+--Ndelta- dipole toward the reduced form of the cluster.     

Kinetic studies of iron deposition in horse spleen ferritin using H2O2 and O2 as oxidants

The reaction of horse spleen ferritin (HoSF) with Fe2+ at pH 6.5 and 7.5 using O2, H2O2 and 1:1 a mixture of both showed that the iron deposition reaction using H2O2 is approximately 20- to 50-fold faster than the reaction with O2 alone. When H2O2 was added during the iron deposition reaction initiated with O2 as oxidant, Fe2+ was preferentially oxidized by H2O2, consistent with the above kinetic measurements. Both the O2 and H2O2 reactions were well defined from 15 to 40 degrees C from which activation parameters were determined. The iron deposition reaction was also studied using O2 as oxidant in the presence and absence of catalase using both stopped-flow and pumped-flow measurements. The presence of catalase decreased the rate of iron deposition by approximately 1.5-fold, and gave slightly smaller absorbance changes than in its absence. From the rate constants for the O2 (0.044 s(-1)) and H2O2 (0.67 s(-1)) iron-deposition reactions at pH 7.5, simulations of steady-state H2O2 concentrations were computed to be 0.45 microM. This low value and reported Fe2+/O2 values of 2.0-2.5 are consistent with H2O2 rapidly reacting by an alternate but unidentified pathway involving a system component such as the protein shell or the mineral core as previously postulated [Biochemistry 22 (1983) 876; Biochemistry 40 (2001) 10832].     

A conserved histidine in vertebrate-type ferredoxins is critical for redox-dependent dynamics

Adrenodoxin (Adx) belongs to the family of Cys(4)Fe(2)S(2) vertebrate-type ferredoxins that shuttle electrons from NAD(P)H-dependent reductases to cytochrome P450 enzymes. The vertebrate-type ferredoxins contain a conserved basic residue, usually a histidine, adjacent to the third cysteine ligand of the Cys(4)Fe(2)S(2) cluster. In bovine Adx the side chain of this residue, His 56, is involved in a hydrogen-bonding network within the domain of Adx that interacts with redox partners. It has been proposed that this network acts as a mechanical link between the metal cluster binding site and the interaction domain, transmitting redox-dependent conformational or dynamical changes from the cluster binding loop to the interaction domain. H/D exchange studies indicate that oxidized Adx (Adx(o)) is more dynamic than reduced Adx (Adx(r)) on the kilosecond time scale in many regions of the protein, including the interaction domain. Dynamical differences on picosecond to nanosecond time scales between the oxidized (Adx(o)) and reduced (Adx(r)) adrenodoxin were probed by measurement of (15)N relaxation parameters. Significant differences between (15)N R(2) rates were observed for all residues that could be measured, with those rates being faster in Adx(o) than in Adx(r). Two mutations of His 56, H56R and H56Q, were also characterized. No systematic redox-dependent differences between (15)N R(2) rates or H/D exchange rates were observed in either mutant, indicating that His 56 is required for the redox-dependent behavior observed in WT Adx. Comparison of chemical shift differences between oxidized and reduced H56Q and H56R Adx confirms that redox-dependent changes are smaller in these mutants than in the wild-type Adx.     

Copper K-extended x-ray absorption fine structure studies of oxidized and reduced dopamine beta-hydroxylase. Confirmation of a sulfur ligand to copper(I) in the reduced enzyme.

The structure of the copper sites in oxidized and reduced dopamine beta-hydroxylase has been studied by extended x-ray absorption fine structure spectroscopy using a restrained refinement approach to data analysis. An histidine-rich active site has been found to be present with an average histidine coordination of between two and three histidine ligands per copper. In the oxidized protein, the data support four-coordination, involving two to three imidazole groups at 1.99 A with additional ligands derived from water or exogenous O-donor groups at an average distance of 1.94 A. Studies on the reduced enzyme have focused on resolving the controversy in the literature (Scott, R. A., Sullivan, R. J., De Wolfe, W. E., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417; Blumberg, W. E., Desai, P. R., Powers, L., Freedman, J. H., and Villafranca, J. J. (1989) J. Biol. Chem. 264, 6029-6032) as to whether a S/Cl scatterer is a ligand to Cu(I). Five independent samples of reduced enzyme prepared under conditions designed to probe the Cu(I) ligand environment have been measured and analyzed. All five samples gave identical spectra and could be simulated by two to three imidazoles (1.93 A) and 0.5 S/Cl (2.25 A) per Cu(I). The spectra were insensitive to the presence of added bromide or to exclusion of chloride during preparation. The results establish that the heavy atom scatterer is derived from a sulfur donor. Some evidence was found for an additional O/N scatterer at 2.6 A in the reduced enzyme. A hypothesis for the structure of the copper sites has been proposed involving inequivalent CuA(His)3(H2O)...CuB-(His)2X(H2O) coordination in the oxidized enzyme, which upon reduction loses coordinated water and coordinates a sulfur probably from a methionine.     

Spectroscopic characterization of cytochrome P450 Compound I

The cytochrome P450 protein-bound porphyrin complex with the iron-coordinated active oxygen atom as Fe(IV)O is called Compound I (Cpd I). Cpd I is the intermediate species proposed to hydroxylate directly the inert carbon–hydrogen bonds of P450 substrates. In the natural reaction cycle of cytochrome P450 Cpd I has not yet been detected, presumably because it is very short-lived. A great variety of experimental approaches has been applied to produce Cpd I artificially aiming to characterize its electronic structure with spectroscopic techniques. In spite of these attempts, none of the spectroscopic studies of the last decades proved capable of univocally identifying the electronic state of P450 Cpd I. Very recently, however, Rittle and Green [9] have shown that Cpd I of CYP119, the thermophillic P450 from Sulfolobus acidocaldarius, is univocally a Fe(IV)O–porphyrin radical with the ferryl iron spin (S = 1) antiferromagnetically coupled to the porphyrin radical spin (S′ = 1/2) yielding a S_tot = 1/2 ground state very similar to Cpd I of chloroperoxidase from Caldariomyces fumago. In this mini-review the efforts to characterize Cpd I of cytochrome P450 by spectroscopic methods are summarized.     

UV Raman monitoring of histidine protonation and H-(2)H exchange in plastocyanin

UV resonance Raman bands of Cu-bound and protonated histidine residues have been detected in (2)H(2)O solutions of poplar plastocyanin. For the Cu(II) protein, slow NH-(2)H exchange of the His37 ligand was monitored via the growth of bands at 1389 and 1344 cm(-1) when Pcy was exchanged into (2)H(2)O, or via their diminution when the protein was exchanged back into H(2)O; the rate constant is 7 x 10(-4)/s at pH (p(2)H) 7.4 at room temperature. The slow exchange is attributed to imidazole H-bonding to a backbone carbonyl. Nearby bands at 1397 and 1354 cm(-1), appear and disappear within the mixing time, and are assigned to the solvent-exposed His87 ligand. The approximately 10 cm(-1) differences between His37 and His87 are attributed to the effect of H-bonding on the imidazole ring modes. The UVRR spectra of the Cu(I) protein in (2)H(2)O reveal a 1408 cm(-1) band, characteristic of NH-(2)H-exchanged histidinium, which grows in as the p(2)H is lowered. Its intensity follows a titration curve with pK(a)=4.6. This protonation is assigned to the His87 residue, whose bond to the Cu(I) is known from crystallography to be broken at low pH. As the 1408 cm(-1) band grows, a band at 1345 cm(-1) diminishes, while another, at 1337 cm(-1) stays constant. These are assigned to modes of bound His87 and His37, respectively, shifted down 7-9 cm(-1) from their Cu(II) positions.     

The first direct characterization of a high-valent iron intermediate in the reaction of an alpha-ketoglutarate-dependent dioxygenase: a high-spin FeIV complex in taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli

The Fe(II)- and alpha-ketoglutarate(alphaKG)-dependent dioxygenases have roles in synthesis of collagen and sensing of oxygen in mammals, in acquisition of nutrients and synthesis of antibiotics in microbes, and in repair of alkylated DNA in both. A consensus mechanism for these enzymes, involving (i) addition of O(2) to a five-coordinate, (His)(2)(Asp)-facially coordinated Fe(II) center to which alphaKG is also bound via its C-1 carboxylate and ketone oxygen; (ii) attack of the uncoordinated oxygen of the bound O(2) on the ketone carbonyl of alphaKG to form a bicyclic Fe(IV)-peroxyhemiketal complex; (iii) decarboxylation of this complex concomitantly with formation of an oxo-ferryl (Fe(IV)=O(2)(-)) intermediate; and (iv) hydroxylation of the substrate by the Fe(IV)=O(2)(-) complex via a substrate radical intermediate, has repeatedly been proposed, but none of the postulated intermediates occurring after addition of O(2) has ever been detected. In this work, an oxidized Fe intermediate in the reaction of one of these enzymes, taurine/alpha-ketoglutarate dioxygenase (TauD) from Escherichia coli, has been directly demonstrated by rapid kinetic and spectroscopic methods. Characterization of the intermediate and its one-electron-reduced form (obtained by low-temperature gamma-radiolysis of the trapped intermediate) by M?ssbauer and electron paramagnetic resonance spectroscopies establishes that it is a high-spin, formally Fe(IV) complex. Its M?ssbauer isomer shift is, however, significantly greater than those of other known Fe(IV) complexes, suggesting that the iron ligands in the TauD intermediate confer significant Fe(III) character to the high-valent site by strong electron donation. The properties of the complex and previous results on related alphaKG-dependent dioxygenases and other non-heme-Fe(II)-dependent, O(2)-activating enzymes suggest that the TauD intermediate is most probably either the Fe(IV)-peroxyhemiketal complex or the taurine-hydroxylating Fe(IV)=O(2)(-) species. The detection of this intermediate sets the stage for a more detailed dissection of the TauD reaction mechanism than has previously been reported for any other member of this important enzyme family.     

Superoxide reductase from Desulfoarculus baarsii: identification of protonation steps in the enzymatic mechanism

Superoxide reductase (SOR) is a metalloenzyme that catalyzes the reduction of O2*- to H2O2 and provides an antioxidant mechanism in some anaerobic and microaerophilic bacteria. Its active site contains an unusual mononuclear ferrous center (center II). Protonation processes are essential for the reaction catalyzed by SOR, since two protons are required for the formation of H2O2. We have investigated the acido-basic and pH dependence of the redox properties of the active site of SOR from Desulfoarculus baarsii, both in the absence and in the presence of O2*-. In the absence of O2*-, the reduction potential and the absorption spectrum of the iron center II exhibit a pH transition. This is consistent with the presence of a base (BH) in close proximity to the iron center which modulates its reduction properties. Studies of mutants of the closest charged residues to the iron center II (E47A and K48I) show that neither of these residues are the base responsible for the pH transitions. However, they both interact with this base and modulate its pKa value. By pulse radiolysis, we confirm that the reaction of SOR with O2*- involves two reaction intermediates that were characterized by their absorption spectra. The precise step of the catalytic cycle in which one protonation takes place was identified. The formation of the first reaction intermediate, from a bimolecular reaction of SOR with O2*-, does not involve proton transfer as a rate-limiting step, since the rate constant k1 does not vary between pH 5 and pH 9.5. On the other hand, the rate constant k2 for the formation of the second reaction intermediate is proportional to the H+ concentration in solution, suggesting that the proton arises directly from the solvent. In fact, BH, E47, and K48 have no role in this step. This is consistent with the first intermediate being an iron(III)-peroxo species and the second one being an iron(III)-hydroperoxo species. We propose that BH may be involved in the second protonation process corresponding to the release of H2O2 from the iron(III)-hydroperoxo species.