Model compounds for polymeric redox-switchable hemilabile ligands

The syntheses and characterization of two new tetradentate hemilabile ligands 1,2-bis(2-diphenylphosphinoethoxy)benzene (5) and 2,2′-bis(2-diphenylphosphinoethoxy)-1,1′-binaphthalene (10) are reported. Ligands 5 and 10 were synthesized as models to test the suitability of specific phosphinoether coordination environments for complexing Rh(I) in high surface area thiophene-based, redox-active polymeric systems. Ligands 5 and 10 react with the product formed from the reaction between (bicyclo[2.2.1]hepta-2,5-diene)rhodium(I) chloride dimer and AgBF₄ to form [η²-(1,2-bis(2-diphenylphosphinoethoxy)benzene) η⁴-norbornadiene rhodium(I)] tetrafluoroborate (6) and [η²-(2,2′-bis(2-diphenylphosphinoethoxy)-1,1′-binaphthalene) η4-norbornadiene rhodium(I)] tetrafluoroborate (11), respectively. Complexes 6 and 11 react with H₂ in CD₂Cl₂ to form the two new square-planar cis-phosphine, cis-ether Rh(I) complexes 7 and 12, respectively. Compound 7, which could be characterized on the basis of its ³¹P NMR spectrum, is extremely reactive and decomposes in CD₂Cl₂. In THF compounds 6 and 11 react with H₂ to form the dihydride, bis-THF adducts 8 and 16, respectively, which upon removal of solvent form 7 and 12, respectively. Compound 12 is a stable, isolable complex that reacts with acetonitrile to form a cis-phosphine, cis-acetonitrile adduct 15. Removal of solvent from 15 leads to the quantitative reformation of 12. Compound 12 does not react to a detectable extent with gross excesses of benzene or even thiophene, demonstrating the suitability of this ligand environment for implementation into a thiophene-based polymeric system. Compound 12 does catalyze the hydrogenation of cyclohexene to form cyclohexane, and mechanistic implications of such a transformation are discussed.     

The hemoglobin of the crossopterygian fish, Latimeria chalumnae (Smith). Subunit structure and oxygen equilibria

There are five oxidation-reduction states of horseradish peroxidase which are interconvertible. These states are ferrous, ferric, Compound II (ferryl), Compound I (primary compound of peroxidase and H₂O₂), and Compound III (oxy-ferrous). The presence of heme-linked ionization groups was confirmed in the ferrous enzyme by spectrophotometric and pH stat titration experiments. The values of pK were 5.87 for isoenzyme A and 7.17 for isoenzymes (B + C). The proton was released when the ferrous enzyme was oxidized to the ferric enzyme while the uptake of the proton occurred when the ferrous enzyme reacted with oxygen to form Compound III. The results could be explained by assuming that the heme-linked ionization group is in the vicinity of the sixth ligand and forms a stable hydrogen bond with the ligand.The measurements of uptake and release of protons in various reactions also yielded the following stoichiometries: Ferric peroxidase + H₂O₂ → Compound I, Compound I + e^? + H⁺ → Compound II, Compound II + e^? + H⁺ → ferric peroxidase, Compound II + H₂O₂ → Compound III, Compound III + 3e^? + 3H⁺ → ferric peroxidase.Based on the above stoichiometries and assuming the interaction between the sixth ligand and heme-linked ionization group of the protein, it was possible to picture simple models showing structural relations between five oxidation-reduction states of peroxidase. Tentative formulae are as follows: [Pr·Po·Fe-(II) $\text{\$}\text{?}$PrH⁺·Po·Fe(II)] is for the ferrous enzyme, Pr·Po·Fe(III)OH₂ for the ferric one, Pr·Po·Fe(IV)OH^? for Compound II, Pr(OH^?)·Po⁺·Fe(IV)OH^? for Compound I, and PrH⁺·Po·Fe(III)O₂^? for Compound III, in which Pr stands for protein and Po for porphyrin. And by Fe(IV)OH^?, for instance, is meant that OH^? is coordinated at the sixth position of the heme iron and the formal oxidation state of the iron is four.     

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.     

Inhibition of sterol biosynthesis by 14α-hydroxymethyl sterols

14α-Hydroxymethyl-5α-cholest-7-en-3β-ol (I) and 14α-hydroxymethyl-5α-cholest-6-en-3β-ol (II) have been prepared by chemical synthesis from 3β-acetoxy-7α,32-epoxy-14α-methyl-5α-cholestane. Compound I, previously shown to be efficiently convertible to cholesterol upon incubation with rat liver homogenate preparations, has been found to be a potent inhibitor of sterol synthesis in animal cells in culture. Compound I caused a 50% reduction of the levels of HMG-CoA reductase activity in cultures of L cells and fetal liver cells at concentrations of 3 × 10^?6 M and 8 × 10^?6 M, respectively. Compound II, the Δ⁶-analogue of I, caused a 50% suppression of the enzyme activity in the two cell types at even lower concentrations, 5 × 10^?7 M and 2 × 10^?6 M, respectively. Concentrations of I and II required to specifically inhibit sterol synthesis from acetate were similar to those required to suppress the levels of HMG-CoA reductase activity.     

Complexity in organic evolution

The present state of knowledge of the formation of the Compounds I of peroxidases and catalases is discussed in terms of the restrictions which must be placed upon a valid mechanism. It is likely that all Compounds I contain one oxygen atom bound to the heme-iron as in the Compound I of chloroperoxidase. Thus the formation of Compound I, obtained after molecular hydrogen peroxide and the enzyme diffuse together, involves a minimum of two bond ruptures and the formation of two new bonds. Yet this amazing reaction proceeds with an activation energy equal to or less than that for the fluidity of water. This result can only be accounted for by including at least one reversible step. Since Compound I formation requires the formation of an “inner sphere” complex, the presence or absence of water in the sixth co-ordination position of the heme-iron is of crucial importance. A comparison of the rates of ligand binding with the rate of Compound I formation indicate that the inner sphere complex leading to Compound I formation is formed by an excellent nucleophile, probably the peroxide anion, formed by a proton transfer from hydrogen peroxide. This proton cannot equilibrate with the bulk solvent. A proton derived from the active site would appear to be added to the hydroxide ion which permits a molecule of water to depart upon oxygen atom addition (or substitution) to (or at) the heme-iron. It is tentatively suggested that Compound I of catalase has a single active site per subunit molecule and that Compound I of peroxidase normally has two reactive sites.     

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.     

Synthesis of phosphonate analogs of sphingomyelins and preparation of an affinity sorbent for sphingomyelinase purification

Phosphonate analogues of 2-N-stearoyl- (I) and 2-N-(undec-10-enoyl)-sphingomyelins (II) have been synthesised. Compound (II) was used as a starting product for preparation of a sorbent for sphingomyelinase affinity chromatography. The double bond of the unsaturated undec-10-enoyl moiety of the phosphonate analogue (II) was oxidized, and the modified (II) was coupled to amino-Toyopearl HW-65 to give a sorbent containing 4 mumoles of ligand per milliliter of the swollen resin.     

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.     

On the structure-function relationship of peroxidases and peroxygenase

The structure-function relationship of two kinds of hemoproteins, peroxidases and peroxygenase, is discussed and a tentative model for the active site (heme vicinity) structure of each hemoprotein is proposed. The mechanism of Compound I formation from peroxidases is presumed to involve an electrophilic attack of hydroperoxide, the electrophilicity of which is increased by forming a hydrogen bond to a distal acid group (with β-equatorial arrangement) on the heme iron, the basicity of which is being increased by electron donation from the anionic fifth ligand. On the other hand, the mechanism for peroxygenase is presumed to involve a nucleophilic attack of hydroperoxide, the nucleophilicity of which is increased by forming a hydrogen bond to a distal base group (with α-axial arrangement) to the heme iron ligating the neutral fifth ligand. It is presumed that Compound I of peroxidases, which consists of porphyrin π cation radical and ferryl iron, is stabilized by a π-π type charge transfer interaction between the radical, and stacking imidazolate group (not necessarily different from the distal group) which then ionizes, and by electron donation from the anionic fifth ligand. On the other hand, Compound I of peroxygenase, which is postulated to be an oxene complex, is presumed to be stabilized by an electrostatic interaction with a strongly negative environment, and by ionization of the fifth ligand, if such can happen.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Selective inhibition by secosteroids of 5 alpha-reductase activity in human sex skin fibroblasts.

The effects of 5,10-secoestra-4,5-diene-3,10,17-trione (Compound I) and 5,10-seco-19-norpregna-4,5-diene,3,10,20-trione (Compound II) on the 5 alpha-reductase activity and on the androgen receptors of normal human sex skin fibroblasts were investigated. The Vmax and Km of the transformation of testosterone to 5 alpha-reduced products was 387 pg/microgram DNA/30 min and 234 X 10(-9)M, respectively. When the inhibitors were introduced in the assay, the 5 alpha-reductase activity was markedly reduced, Compound I being a less potent inhibitor than Compound II. At 15 min, the inhibition was greater than at 30 and 60 min. The Ki for Compound I was 1.60 x 10(-6)M with a Vmax of 83 to 553 pg/microgram DNA/30 min. For Compound II, the Ki was 0.53 x 10(-6)M with a Vmax of 70 to 340 pg/microgram DNA/30 min. The inhibition was of the noncompetitive type. Studies with androgen receptors showed that Compound I had a lower affinity for the receptors than Compound II. The ID50 for 3H-DHT and 3H-T for Compound I were 42.9 x 10(-7)M and 8.6 x 10(-7)M, respectively, whereas for Compound II, they were 10.6 x 10(-7)M and 4.8 x 10(-7)M.     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

The properties of the secondary catalase—peroxide complex (Compound II) in the hemoglobin-free perfused rat liver

The possibility of the occurrence of the secondary catalase-peroxide complex (Compound II) in the isolated peroxisomal-mitochondrial fraction of rat liver and in the perfused rat liver has been examined under various conditions. The steady state of Compound I is maintained by either an endogeneous or a urate and glycolate-supplemented H₂O₂ generation in both systems, but Compound II is not detectable. Significant accumulation of Compound II, which is identified by the measurement of its difference spectrum and by its response to hydrogen donors, is observed only when Compound I is converted to Compound II by an appropriate concentration of p-cresol. The properties of Compound II observed in the perfused liver are similar to those observed with isolated catalase.     

Delocalization over the heme and the axial ligands of one of the two oxidizing equivalents stored above the ferric state in the peroxidase and catalase Compound-I intermediates: indirect participation of the proximal axial ligand of iron in the oxidation reactions catalyzed by heme-based peroxidases and catalases?

 A comparison of the exchange interactions arising in the peroxidase and catalase Compound I intermediates and their iron(IV)-oxo porphyrin π-cation radical models, both of which are two oxidizing equivalents above the ferric state, suggests that in the models the oxidizing equivalent is localized on the porphyrin ring, while in the proteins it is partly delocalized onto the proximal ligand. Thus, the proximal axial ligand of iron participates indirectly in the oxidation reactions catalyzed by the enzymes. Possible roles of the axial ligand in the catalytic mechanism of these heme-based enzymes are discussed. Received and accepted: 7 May 1996     

Comparative electron paramagnetic resonance study of radical intermediates in turnip peroxidase isozymes

The occurrence of isozymes in plant peroxidases is poorly understood. Turnip roots contain seven season-dependent isoperoxidases with distinct physicochemical properties. In the work presented here, multifrequency electron paramagnetic resonance spectroscopy has been used to characterize the Compound I intermediate obtained by the reaction of turnip isoperoxidases 1, 3, and 7 with hydrogen peroxide. The broad (2500 G) Compound I EPR spectrum of all three peroxidases was consistent with the formation of an exchange-coupled oxoferryl-porphyrinyl radical species. A dramatic pH dependence of the exchange interaction of the [Fe(IV)=O por(*+)] intermediate was observed for all three isoperoxidases and for a pH range of 4.5-7.7. This result provides substantial experimental evidence for previous proposals concerning the protein effect on the ferro- or antiferromagnetic character of the exchange coupling of Compound I based on model complexes. Turnip isoperoxidase 7 exhibited an unexpected pH effect related to the nature of the Compound I radical. At basic pH, a narrow radical species ( approximately 50 G) was formed together with the porphyrinyl radical. The g anisotropy of the narrow radical Delta(g) = 0.0046, obtained from the high-field (190 and 285 GHz) EPR spectrum, was that expected for tyrosyl radicals. The broad g(x) edge of the Tyr* spectrum centered at a low g(x) value (2.00660) strongly argues for a hydrogen-bonded tyrosyl radical in a heterogeneous microenvironment. The relationship between tyrosyl radical formation and the higher redox potential of turnip isozyme 7, as compared to that of isozyme 1, is discussed.     

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.     

The reaction of Aspergillus niger catalase with methyl hydroperoxide.

The formation of Compound I from Aspergillus niger catalase and methyl hydroperoxide (CH3OOH) has been investigated kinetically by means of rapid-scanning stopped-flow techniques. The spectral changes during the reaction showed distinct isobestic points. The second-order rate constant and the activation energy for the formation of Compound I were 6.4 x 10(3) M-1s-1 and 10.4 kcal.mol-1, respectively. After formation of Compound I, the absorbance at the Soret peak returned slowly to the level of ferric enzyme with a first-order rate constant of 1.7 x 10(-3) s-1. Spectrophotometric titration of the enzyme with CH3OOH indicates that 4 mol of peroxide react with 1 mol of enzyme to form 1 mol of Compound I. The amount of Compound I formed was proportional to the specific activity of the catalase. The irreversible inhibition of catalase by 3-amino-1,2,4-triazole (AT) was observed in the presence of CH3OOH or H2O2. The second-order rate constant of the catalase-AT formation in CH3OOH was 3.0 M-1 min-1 at 37 degrees C and pH 6.8 and the pKa value was estimated to be 6.10 from the pH profile of the rate constant of the AT-inhibition. These results indicate that A. niger catalase forms Compound I with the same properties as other catalases and peroxidases, but the velocity of the Compound I formation is lower than that of the others.     

Studies on Phyto-peroxidase

Japanese-radish peroxidase c, a paraperoxidase exhibiting the optical absorption spectrum of low-spin nature, was found to transform to a high-spin state by removing a dissociable ligand of low molecular weight by the addition of the stoichiometric amount of p-chloro mercuribenzoate, as in the case of horseradish peroxidase I or wheat germ peroxidase 566. The reaction could be reversed by the addition of cysteine to remove p-chloromercuribenzoate. As this ligand would be possibly cyanide, the affinity of the high-spin form of the enzyme to sodium cyanide was determined, which was found to be much higher than that of Japanese-radish peroxidase a. The high-spin form of peroxidase c formed the usual Compound I by the addition of hydrogen peroxide, so that the peroxidatic reaction catalyzed by this enzyme should follow the common mechanism of plant peroxidases. However, Compound II was scarecely observed during the course of the stepwise reduction of Compound I by ascorbate, probably because of its more rapid conversion to the free enzyme.