Dioxygen activation at mononuclear nonheme iron active sites: enzymes, models, and intermediates.

The p38 signal transduction pathway: activation and function.

ion step follows the decarboxylation, which is consistent with the deuterium isotopic effects observed for thymine 7-hydroxylase which indicate that an irreversible step (or steps) occurs prior to the C-H bond breaking.395 It has also been shown for prolyl 4-hydroxylase that a substrate-derived radical is generated in the reaction, which is consistent with a rebound mechanism.437 It is important to point out that no oxygen intermediate (i.e., bridged superoxo or oxo-ferryl) has been observed for any R-KGdependent enzyme. This warrants future theoretical and experimental study. A detailed molecular mechanism has been proposed for IPNS based on spectroscopic and crystallographic studies.422 Resting IPNS/FeII is also 6C and thus relatively stable toward dioxygen. Substrate ACV binds directly to FeII IPNS through its thiolate group, providing an open coordination position at the FeII. O2 can then react to form an FeIII-superoxo intermediate. This intermediate is suggested422 to perform the first hydrogen-atom abstraction step and close the â-lactam ring, resulting in the formation of the first water molecule and generating an FeIVdO-II intermediate, which completes the second ringclosure process by hydrogen-atom abstraction forming a thiazolidine ring. Previously proposed mechanisms of ACCO involved direct binding of cosubstrate ascorbate to the iron before O2 as part of the oxygen activation process.438,439 The EPR and ESEEM studies of the NO complex of ACCO suggested a quite different molecular mechanism for ACCO.435 An FeIII-superoxo intermediate is proposed. Whether it is preceded by a 6C f 5C process with substrate binding is presently under study.440 This intermediate is thought to initiate a radical process by single hydrogen-atom abstraction or electron-coupled proton transfer (PT)ion or electron-coupled proton transfer (PT) from the bound amino group. The resulting substrate radical may undergo spontaneous conversion into products. The role of cosubstrate ascorbate is proposed to reduce the toxic peroxo byproduct to water. Alternatively, the two-electron reduction of FeIIIsuperoxo by the cosubstrate ascorbate could result in an FeIVdO-II intermediate which initiates the radical reaction.435 4. Rieske-Type Dioxygenases Biochemical Characterization. The Rieske ironsulfur center is a two iron-two sulfur cluster ([2Fe2S]) which has a 2His (on one iron), 2Cys (on the other iron) coordination environment, instead of the 4Cys present in plant ferredoxins. It plays a key role in the electron transport pathway in membranebound cytochrome complexes as well as in some dioxygenases.441 The latter are mainly comprised of two protein components: a reductase containing flavin and a ferredoxin [2Fe-2S], and a terminal oxygenase containing a Rieske [2Fe-2S] cluster and a non-heme iron active site.442 Except for the recently reported alkene monooxygenase that has a binuclear iron site in its terminal oxygenase,10 most of the Rieske-type oxygenases have a mononuclear iron site, which is believed to be the site of dioxygen activation and substrate oxygenation.442,443 The majority of the Rieske-type mononuclear non-heme oxygenases form a family of enzymes which are aromatic-ring-hydroxylating dioxygenases. These catalyze the regioand stereospecific cis-dihydroxylation of an aromatic ring using dioxygen and NAD(P)H (Table 1). Examples include benzene dioxygenase (BDO, EC 1.14.12.3),444 phthalate dioxygenase (PDO, EC 1.14.12.7),445 toluene dioxygenase (EC 1.14.12.11),446 and naphthalene 1,2-dioxygenase (NDO, EC 1.14.12.12),447 which initiate the aerobic degradation of aromatic compounds in the soil bacteria and are targets for bioengineering in bioremediation. This step is the first step in the pathway that ultimately leads to ring cleavage by the intraand extradiol dioxygenases (sections II.B.2 and II.C.1).443 Besides these bacterial dioxygenases, other Rieske-type mononuclear non-heme oxygenases include anthranilate 1,2-dioxygenase (EC 1.14.12.1),448 which deaminates and decarboxylates the substrate to produce catechol; chlorophenylacetate 3,4-dioxygenase (EC 1.14.2.13),449 which converts substrate to catechol with chloride elimination; and 4-methoxybenzoate O-demethylase (putidamonooxin),450 which catalyzes the conversion of 4-methoxybenzoic acid to 4-hydroxybenzoic acid and formaldehyde. The reductase component is usually a monomer (MW ) 12-15 kDa) and utilizes flavin to mediate ET from the two-electron donor NAD(P)H to the oneelectron acceptor [2Fe-2S] cluster and is specific to each terminal oxygenase; other electron donors do not support efficient oxygenation.442 The crystal structure of phthalate dioxygenase reductase is available.451 The terminal oxygenases are large protein aggregates (MW ) 150-200 kDa) containing either multiples of R subunits (BDO R2, PDO R4) or an equimolar combination of R and â subunits (toluene dioxygenase R2â2, NDO R3â3). The R subunits contain a Rieske [2Fe-2S] cluster and a catalytic non-heme FeII center. â subunits do not seem to be involved in the catalytic function (vide infra). Kinetics. Steady-state kinetic studies coupled with various rapid reaction studies of the partial reactions of PDO allowed Ballou et al. to propose a kinetic scheme (Scheme 15).443 On the basis of steady state 278 Chemical Reviews, 2000, Vol. 100, No. 1 Solomon et al.

Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes

The expanding superfamily of phospholipase A(2) enzymes: classification and characterization.

X-ray absorption Fe−K edge data on ferrous and ferric model complexes have been studied to establish a detailed understanding of the 1s → 3d pre-edge feature and its sensitivity to the electronic structure of the iron site. The energy position and splitting, and intensity distribution, of the pre-edge feature were found to vary systematically with spin state, oxidation state, geometry, and bridging ligation (for binuclear complexes). A methodology for interpreting the energy splitting and intensity distribution of the 1s → 3d pre-edge features was developed for high-spin ferrous and ferric complexes in octahedral, tetrahedral, and square pyramidal environments and low-spin ferrous and ferric complexes in octahedral environments. In each case, the allowable many-electron excited states were determined using ligand field theory. The energies of the excited states were calculated and compared to the energy splitting in the 1s → 3d pre-edge features. The relative intensities of electric quadrupole transitions...

A Multiplet Analysis of Fe K-Edge 1s → 3d Pre-Edge Features of Iron Complexes

X-ray absorption spectroscopy of iron-tyrosinate proteins

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Previous studies using phospholipid mixed vesicles have demonstrated that several types of phospholipase A2 (PLA2) enzymes exhibit different selectivity for fatty acids at the sn-2 position, for the type of chemical bond at the sn-1 position or for the phosphobase moiety at the sn-3 position of phospholipids. In the present study, we have utilized natural mammalian membranes from U937 monocytes to determine whether two purified 14 kDa PLA2 isoenzymes (Type I, Type II) and a partially purified 110 kDa PLA2 exhibit substrate selectivity for certain fatty acids or phospholipids. In these studies, arachidonic acid (AA) release from membranes was measured under conditions where the remodelling of AA mediated by CoA-independent transacylase (CoA-IT) activity has been eliminated. In agreement with the mixed-vesicle models, AA was the major unsaturated fatty acid hydrolysed from membranes by the 110 kDa PLA2, suggesting that this PLA2 is selective in releasing AA from natural membranes. By contrast, Type I and Type II PLA2s were less selective in releasing AA from phospholipids and released a variety of unsaturated fatty acids at molar ratios that were proportional to the ratios of these fatty acids in U937 microsomal membranes. Examination of AA release from phospholipid classes indicated that all three enzymes released AA from the major AA-containing phospholipid classes (phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol) of U937 membranes. The 110 kDa PLA2 released AA from phospholipid subclasses in ratios that were proportional to the AA content within phospholipid classes and subclasses of U937 membranes. These data suggested that the 110 kDa PLA2 shows no preference either for the sn-1 linkage or for the sn-3 phosphobase moiety of phospholipids. By contrast, Type I and Type II PLA2s preferentially released AA from ethanolamine-containing phospholipids and appeared to prefer the 1-acyl-linked subclass. Taken together, these data indicate that the 110 kDa PLA2 selectively releases AA from U937 membranes, whereas Type I and Type II PLA2 release a variety of unsaturated fatty acids. Furthermore, the 110 kDa PLA2 releases the same molar ratios of AA from all major phospholipid subclasses, whereas Type I and Type II PLA2s show some specificity for phosphatidylethanolamine when these enzymes are incubated with a complex mammalian membrane substrate.

Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate

Rapid reaction studies on the oxygenation reactions of catechol dioxygenase.

The resonance Raman spectra of native pyrocatechase and its benzoate and phenolate complexes were investigated by using the available lines of an argon and a krypton laser. The data provide evidence for the presence of two distinct tyrosines coordinated to the active-site iron. The two tyrosines exhibit different upsilon CO values which show maximum resonance enhancements at different excitation wavelengths. Moreover, one tyrosine is more susceptible to changes in the active-site environment. Pyrocatechase is the only example thus far among iron-tyrosinate proteins where the tyrosines coordinating the iron are distinguishable.

Ruth J. Mayer

Papers

X-ray absorption spectroscopy of iron-tyrosinate proteins

Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model.

Fatty acid and phospholipid selectivity of different phospholipase A2 enzymes studied by using a mammalian membrane as substrate

Rapid reaction studies on the oxygenation reactions of catechol dioxygenase.

Resonance Raman studies of pyrocatechase-inhibitor complexes.