Theoretical studies of enzyme mechanisms involving high-valent iron intermediates

Recent theoretical contributions to the elucidation of mechanisms for iron containing enzymes are reviewed. The method used in most of these studies is hybrid density functional theory with the B3LYP functional. Three classes of enzymes are considered, the mononuclear non-heme enzymes, enzymes containing iron dimers, and heme-containing enzymes. Mechanisms for both dioxygen and substrate activations are discussed. The reactions usually go through two half-cycles, where a high-valent intermediate Fe(IV)O species is created in the first half-cycle, and the substrate reactions involving this intermediate occur in the second half-cycle. Similarities between the three classes of enzymes dominate, but significant differences also exist.     

A role for rubredoxin in oxidative stress protection in Desulfovibrio vulgaris: catalytic electron transfer to rubrerythrin and two-iron superoxide reductase

Desulfovibrio vulgaris rubredoxin, which contains a single [Fe(SCys)4] site, is shown to be a catalytically competent electron donor to two enzymes from the same organism, namely, rubrerythrin and two-iron superoxide reductase (a.k.a. rubredoxin oxidoreductase or desulfoferrodoxin). These two enzymes have been implicated in catalytic reduction of hydrogen peroxide and superoxide, respectively, during periods of oxidative stress in D. vulgaris, but their proximal electron donors had not been characterized. We further demonstrate the incorrectness of a previous report that rubredoxin is not an electron donor to the superoxide reductase and describe convenient assays for demonstrating the catalytic competence of all three proteins in their respective functions. Rubrerythrin is shown to be an efficient rubredoxin peroxidase in which the rubedoxin:hydrogen peroxide redox stoichiometry is 2:1 mol:mol. Using spinach ferredoxin-NADP+ oxidoreductase (FNR) as an artificial, but proficient, NADPH:rubredoxin reductase, rubredoxin was further found to catalyze rapid and complete reduction of all Fe3+ to Fe2+ in rubrerythrin by NADPH under anaerobic conditions. The combined system, FNR/rubredoxin/rubrerythrin, was shown to function as a catalytically competent NADPH peroxidase. Another small rubredoxin-like D. vulgaris protein, Rdl, could not substitute for rubredoxin as a peroxidase substrate of rubrerythrin. Similarly, D. vulgaris rubredoxin was demonstrated to efficiently catalyze reduction of D. vulgaris two-iron superoxide reductase and, when combined with FNR, to function as an NADPH:superoxide oxidoreductase. We suggest that, during periods of oxidative stress, rubredoxin could divert electron flow from the electron transport chain of D. vulgaris to rubrerythrin and superoxide reductase, thereby simultaneously protecting autoxidizable redox enzymes and lowering intracellular hydrogen peroxide and superoxide levels.     

Valence-tautomerism in high-valent iron and manganese porphyrins

Iron and manganese hemes are "high-valent" when the valence state of the metal exceeds III. Redox chemistry of the high valent metal complexes involves redistribution of holes and electrons over the metal ion and the porphyrin and axial ligands, defined as valence tautomerism. Thus, catalytic pathways of heme-containing biomolecules such as peroxidases, catalases and cytochromes P450 involve valence tautomerism, as do pathways of biomimetic oxygen transfer catalysis by manganese porphyrins, robust catalysts with potential commercial value. Determinants of the site of electron abstraction are key to understanding valence tautomerism. In model systems, metal-centered oxidation is supported by hard anionic axial ligands that are also strongly pi-donating, such as oxo, aryl, bix-methoxy and bis-fluoro groups. Manganese(IV) is more stable than iron(IV) and metal-centered one-electron oxidations occur with weaker pi-donating axial ligands such as bisazido, -isocyanato, -hypochlorito and bis chloro groups. Virtually all known high-valent iron porphyrin complexes oxidized by two-electrons above the ferric state are coordinated by the strongly pi-donating oxo or nitrido ligands. In all well-characterized oxo complexes, iron is in the ferryl state and the second oxidizing equivalent resides on the porphyrin. Complexes with iron(V) have not been definitively characterized. One-electron oxidation of oxomanganese(IV) porphyrin complexes gives the oxomanganese(IV) porphyrin pi-cation redicals. In aqueous solution, oxidation of Mn(III) complexes of tetra cationic N-methylpyridiniumylporphyrin isomers by monooxygen donors yields a transient oxomanganese(V) species.     

Mid- to high-valent imido and nitrido complexes of iron

This short review summarizes recent advances in the synthesis and reactivity of iron imides and nitrides. Both monometallic and multimetallic assemblies featuring these moieties are discussed. Recent synthetic approaches have led to the availability of new mid- to high-valent iron imides and nitrides, allowing us to begin assembling models to describe the factors influencing their relative stabilities and reactivity patterns.     

Structures of the high-valent metal-ion haem-oxygen intermediates in peroxidases, oxygenases and catalases

Peroxidases, oxygenases and catalases have similar high-valent metal-ion intermediates in their respective reaction cycles. In this review, haem-based examples will be discussed. The intermediates of the haem-containing enzymes have been extensively studied for many years by different spectroscopic methods like UV-Vis, EPR (electron paramagnetic resonance), resonance Raman, M?ssbauer and MCD (magnetic circular dichroism). The first crystal structure of one of these high-valent intermediates was on cytochrome c peroxidase in 1987. Since then, structures have appeared for catalases in 1996, 2002, 2003, putatively for cytochrome P450 in 2000, for myoglobin in 2002, for horseradish peroxidase in 2002 and for cytochrome c peroxidase again in 1994 and 2003. This review will focus on the most recent structural investigations for the different intermediates of these proteins. The structures of these intermediates will also be viewed in light of quantum mechanical (QM) calculations on haem models. In particular quantum refinement, which is a combination of QM calculations and crystallography, will be discussed. Only small structural changes accompany the generation of these intermediates. The crystal structures show that the compound I state, with a so called pi-cation radical on the haem group, has a relatively short iron-oxygen bond (1.67-1.76A) in agreement with a double-bond character, while the compound II state or the compound I state with a radical on an amino acid residue have a relatively long iron-oxygen bond (1.86-1.92A) in agreement with a single-bond character where the oxygen-atom is protonated.     

Discovery of superoxide reductase: an historical perspective

For more than 30 years, the only enzymatic system known to catalyze the elimination of superoxide was superoxide dismutase, SOD. SOD has been found in almost all organisms living in the presence of oxygen, including some anaerobic bacteria, supporting the notion that superoxide is a key and general component of oxidative stress. Recently, a new concept in the field of the mechanisms of cellular defense against superoxide has emerged. It was discovered that elimination of superoxide in some anaerobic and microaerophilic bacteria could occur by reduction, a reaction catalyzed by a small metalloenzyme thus named superoxide reductase, SOR. Having played a major role in this discovery, we describe here how the concept of superoxide reduction emerged and how it was experimentally substantiated independently in our laboratory.Abbreviations Dfx desulfoferrodoxin - SOD superoxide dismutase - SOR superoxide reductase     

Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O(2) as well as the reduction of ferricyanide by O(2). Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H(2)O(2) production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.     

Reaction of Desulfovibrio vulgaris two-iron superoxide reductase with superoxide: insights from stopped-flow spectrophotometry

Stopped-flow mixing of the Desulfovibrio vulgaris two-iron superoxide reductase (2Fe-SOR) containing the ferrous active site with superoxide generates a dead time intermediate whose absorption spectrum is identical to that of a putative ferric-hydroperoxo intermediate previously observed by pulse radiolysis. The dead time intermediate is shown to be a product of reaction with superoxide and to be generated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decays smoothly to the resting ferric active site ( approximately 30 s-1 at 2 degrees C and pH 7) with no other detectable intermediates. Deuterium isotope effects demonstrate that solvent proton donation occurs in the rate-determining step of dead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functions as a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphate accelerate decay of the dead time intermediate and for azide or fluoride lead directly to ferric-azido or -fluoro complexes of the active site, which inhibit Glu47 ligation. A solvent deuterium isotope effect is observed for the azide-accelerated decay, and the decay rate constants are proportional to the concentrations and pKa values of HX (X- = F-, HCO2-, N3-). These data indicate that the protonated forms of the anions function analogously to solvent as general acids in the rate-determining step. The results support the notion that the ferrous SOR site reacts with superoxide by an inner sphere process, leading directly to the ferric-hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject to both specific- and general-acid catalysis.     

High-valent transition metal centers versus noninnocent ligands in metallocorroles: insights from electrochemistry and implications for high-valent heme protein intermediates

For relatively electron-rich corrole ligands, the halfwave potentials for oxidation of Cu(III), Sn(IV)Ph, Fe(IV)Ph, and Fe(IV)-O-Fe(IV) complexes are significantly lower than those of Sn(IV)Cl, Fe(IV)Cl, Mn(IV)Cl, and Cr(V)(O) complexes, suggesting that the corrole ligand is relatively electron-rich or 'innocent' in the former group of complexes and that it is relatively electron-deficient or 'noninnocent' in the latter group. Both the formal charge of the central metal ion and the nature of the axial ligand, if any, appear to be key determinants of the electronic character of the corrole ligand in metallocorrole complexes, a theme that has interesting resonances with recent findings on high-valent heme protein intermediates. However, for very strongly electron-deficient ligands such as meso-tris(pentafluorophenyl)corrole (TPFPC) and beta-octabromo-meso-tris(pentafluorophenyl)corrole (Br(8)TPFPC), which cannot sustain significant radical character, the various metal complexes all exhibit comparable halfwave potentials for oxidation and the ligand may be considered to be relatively innocent.     

Kinetics and mechanism of superoxide reduction by two-iron superoxide reductase from Desulfovibrio vulgaris

Superoxide reductases (SORs) contain a novel square pyramidal ferrous [Fe(NHis)(4)(SCys)] site that rapidly reduces superoxide to hydrogen peroxide. Here we report extensive pulse radiolysis studies on recombinant two-iron SOR (2Fe-SOR) from Desulfovibrio vulgaris. The results support and elaborate on our originally proposed scheme for reaction of the [Fe(NHis)(4)(SCys)] site with superoxide [Coulter, E. D., Emerson, J. E., Kurtz, D. M., Jr., and Cabelli, D. E. (2000) J. Am. Chem. Soc. 122, 11555-11556]. This scheme consists of second-order diffusion-controlled formation of an intermediate absorbing at approximately 600 nm, formulated as a ferric-(hydro)peroxo species, and its decay to the carboxylate-ligated ferric [Fe(NHis)(4)(SCys)] site with loss of hydrogen peroxide. The second-order rate constant for formation of the 600-nm intermediate is essentially pH-independent (pH 5-9.5), shows no D(2)O solvent isotope effect at pH 7.7, and decreases with increasing ionic strength. These data indicate that formation of the intermediate does not involve a rate-determining protonation, and are consistent with interaction of the incoming superoxide anion with a positive charge at or near the ferrous [Fe(NHis)(4)(SCys)] site. The rate constant for decay of the 600-nm intermediate follows the pH-dependent rate law: k(2)(obs) = k(2)'[H(+)] + k(2)' ' and shows a significant D(2)O solvent isotope effect at pH 7.7. The values of k(2)' and k(2)' ' indicate that the 600-nm intermediate decays via diffusion-controlled protonation at acidic pHs and a first-order process involving either water or a water-exchangeable proton on the protein at basic pHs. The formation and decay rate constants for an E47A variant of 2Fe-SOR are not significantly perturbed from their wild-type values, indicating that the conserved glutamate carboxylate does not directly displace the (hydro)peroxo ligand of the intermediate at basic pHs. The kinetics of a K48A variant are consistent with participation of the lysyl side chain in directing the superoxide toward the active site and in directing the protonation pathway of the ferric-(hydro)peroxo intermediate toward release of hydrogen peroxide.     

The iron content of iron superoxide dismutase: determination by anomalous scattering

The number of iron atoms in the dimeric iron-containing superoxide dismutase from Pseudomonas ovalis and their atomic positions have been determined directly from anomalous scattering measurements on crystals of the native enzyme. To resolve the long-standing question of the total amount of iron per molecule for this class of dismutase, the occupancy of each site was refined against the measured Bijvoet differences. The enzyme is a symmetrical dimer with one iron site in each subunit. The iron position is 9 A from the intersubunit interface. The total iron content of the dimer is 1.2 +/- 0.2 moles per mole of protein. This is divided between the subunits in the ratio 0.65:0.55; the difference between them is probably not significant. Since each subunit contains, on average, slightly more than half an iron atom we conclude that the normal state of this enzyme is two iron atoms per dimer but that some of the metal is lost during purification of the protein. Although the crystals are obviously a mixture of holo- and apo-enzymes, the 2.9 A electron density map is uniformly clean, even at the iron site. We conclude that the three-dimensional structures of the iron-bound enzyme and the apo-enzyme are identical.     

Substrate activation by iron superoxo intermediates

A growing number of non-heme-iron oxygenases and oxidases catalyze reactions for which the well-established mechanistic paradigm involving a single C-H-bond-cleaving intermediate of the Fe(IV)-oxo (ferryl) type [1(?)] is insufficient to explain the chemistry. It is becoming clear that, in several of these cases, Fe(III)-superoxide complexes formed by simple addition of O(2) to the reduced [Fe(II)] cofactor initiate substrate oxidation by abstracting hydrogen [2,3(?)]. This substrate-oxidizing entry route into high-valent-iron intermediates makes possible an array of complex and elegant oxidation reactions without the consumption of valuable reducing equivalents. Examples of this novel mechanistic strategy are discussed with the goal of bringing forth unifying principles.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

Kinetics of the superoxide reductase catalytic cycle

The steady state kinetics of a Desulfovibrio (D.) vulgaris superoxide reductase (SOR) turnover cycle, in which superoxide is catalytically reduced to hydrogen peroxide at a [Fe(His)4(Cys)] active site, are reported. A proximal electron donor, rubredoxin, was used to supply reducing equivalents from NADPH via ferredoxin: NADP+ oxidoreductase, and xanthine/xanthine oxidase was used to provide a calibrated flux of superoxide. SOR turnover in this system was well coupled, i.e. approximately 2O*2 reduced:NADPH oxidized over a 10-fold range of superoxide flux. The reduction of the ferric SOR active site by reduced rubredoxin was independently measured to have a second-order rate constant of approximately 1 x 10(6) m-1 s-1. Analysis of the kinetics showed that: (i) 1 microM SOR can convert a 10 microM/min superoxide flux to a steady state superoxide concentration of 10(-10) m, during which SOR turns over about once every 6 s, (ii) the diffusion-controlled reaction of reduced SOR with superoxide is the slowest process during turnover, and (iii) neither ligation nor deligation of the active site carboxylate of SOR limits the turnover rate. An intracellular SOR concentration on the order of 10 microM is estimated to be the minimum required for lowering superoxide to sublethal levels in aerobically growing SOD knockout mutants of Escherichia coli. SORs from Desulfovibrio gigas and Treponema pallidum showed similar turnover rates when substituted for the D. vulgaris SOR, whereas superoxide dismutases showed no SOR activity in our assay. These results provide quantitative support for previous suggestions that, in times of oxidative stress, SORs efficiently divert intracellular reducing equivalents to superoxide.     

Resonance Raman characterization of the mononuclear iron active-site vibrations and putative electron transport pathways in Pyrococcus furiosus superoxide reductase

The resonance Raman spectrum of oxidized wild-type P. furiosus SOR at pH 7.5 and 10.5 has been investigated using excitation wavelengths between 406 and 676 nm, and vibrational modes have been assigned on the basis of isotope shifts resulting from global replacements of (32)S with (34)S, (14)N with (15)N, (56)Fe with (54)Fe, and exchange into a H(2)(18)O buffer. The results are interpreted in terms of the crystallographically defined active-site structure involving a six-coordinate mononuclear Fe center with four equatorial histidine ligands and axial cysteine and monodentate glutamate ligands (Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. C. (2000) Biochemistry 39, 2499-2508). Excitation into the intense (Cys)S(p(pi))-to-Fe(d(pi)) CT transition centered at 660 nm results in strong enhancement of modes at 298 cm(-1) and 323 cm(-1) that are assigned to extensively mixed cysteine S-C(beta)-C(alpha) bending and Fe-S(Cys) stretching modes, respectively. All other higher-energy vibrational modes are readily assigned to overtone or combination bands or to fundamentals corresponding to internal modes of the ligated cysteine. Weak enhancement of Fe-N(His) stretching modes is observed in the 200-250 cm(-1) region. The enhancement of internal cysteine modes and Fe-N(His) stretching modes are a consequence of a near-planar Fe-S-C(beta)-C(alpha)-N unit for the coordinated cysteine and significant (His)N(p(pi))-Fe(d(xy))-(Cys)S(p(pi)) orbital overlap, respectively, and have close parallels to type 1 copper proteins. By analogy with type 1 copper proteins, putative superexchange electron-transfer pathways to the mononuclear Fe active site are identified involving either the tyrosine and cysteine residues or the solvent-exposed deltaN histidine residue in a Y-C-X-X-H arrangement. Studies of wild-type at pH 10.5 and the E14A variant indicate that the resonance Raman spectrum is remarkably insensitive to changes in the ligand trans to cysteine and hence are inconclusive concerning the origin of the alkaline transition and the nature of sixth Fe ligand in the E14A variant.     

Phytomonas iron superoxide dismutase: a possible molecular marker

We have isolated and biochemically characterized two iron superoxide dismutases activities (SODI and SODII) from a plant trypanosomatid isolated from Euphorbia characias. The isoenzyme FeSODII has immunogenic capacity, and the positivity of the anti-SODII serum persists to a dilution of 1/40,000, by Western blot. In addition, Western blot has been used to test the positivity of the anti-SODII serum against antigen fractions (SOD) from 17 isolates belonging to the family Trypanosomatidae and for which we had previously determined the isoenzymatic profile. The reaction proved positive only with those plant isolates considered to belong to the genus Phytomonas, whereas there was no reaction of the anti-SODII serum, against the antigen fractions from the species Trypanosoma cruzi, Leishmania donovani, Herpetomonas samuelpessoai, Herpetomonas davidi, Crithidia luciliae and Leptomonas collosoma. FeSODII is located mainly over the entire surface of the parasite, as well as in the nucleus, glycosomes and membranes. The above makes FeSODII promising as a molecular tool for diagnosis and identification, and as a potential chemotherapeutic target for designing drugs aimed at controlling not only of the diseases caused by Phytomonas species, but also for the great metabolic similarity to other trypanosomatids of animals and humans, it may be possible for these results to be extrapolated. Moreover, the sequencing of the amino-terminal end of the FeSODII enables the design of primers that in the near future will make it possible to sequence the gene of this isoenzyme.     

Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster

A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and M?ssbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The M?ssbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)