Iron dependent hydroxylation

McCormick ML, GR Buettner, BE Britigan (1998) Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide. J Bacterial 180 622-625. 

Iron dependent hydroxyl radical formation (assay I). The sample contained the following substances in lmL 50 mmol/L potassium phosphate buffer, pH 7.4 50 pmol/L luminol, 100 pmol/L Fe3+, 100 pmol/L EDTA, 100 pmol/L ascorbate, 500 pmol/L H2O2 and either the tested extract at the concentrations shown in Fig. 1 or a buffer as a control. The CL was measured using the flash assay option of the MultiUse program, every 50 milliseconds. 

The mechanism of iron dependent hydroxylation by RLPAH was compared with that of the metal-independent one by CVPAH to clarify the role of iron in the mechanism of RLPAH [122]. A kinetic isotope effect, / d, on hydroxylation of [4- H]-phenylalanine as a substrate is unity for CVPAH, which is in agreement with values for RLPAH [123]. An NIH shift was observed with the [4- H]-phenylalanine upon hydroxylation by CVPAH as by RLPAH [124-126]. The extent of deuterium retention at the 3-position of the tyrosine product was identical within experimental error for both CVPAH and... 

Tyrosine hydroxylase (TH) is an enzyme that catalyzes the hydroxylation of tyrosine to 3,4-dihydroxypheny-lalanine in the brain and adrenal glands. TH is the rate-limiting enzyme in the biosynthesis of dopamine. This non-heme iron-dependent monoxygenase requires the presence of the cofactor tetrahydrobiopterin to maintain the metal in its ferrous state. 

Halliwell B. Use of desferrioxamine as a probe for iron-dependent formation of hydroxyl radicals. Evidence for a direct reaction between desferal and the superoxide radical. Biochem Pharmacol 1985 34(2) 229-233. 

The ability to synthesise ascorbic acid from glucose is absent in a small group of animal species that include man, primates, the guinea pig and the fruit-bat this is due to the absence of the gene that codes for one of the enzymes required for ascorbate synthesis. These species are therefore dependent on an external source of the vitamin in their diet and it is needed as a cofactor for several hydroxylase enzymes, notably the iron-dependent proline and lysine hydroxylases and the copper-dependent dopamine-(3-hydroxylase the function of ascorbate in these enzymes is likely to be its ability to keep the metal in the reduced form which is necessary for hydroxylation. The ability of ascorbate to reduce Fe3+ to Fe2+ is important in promoting the gastrointestinal uptake of iron and for its release from the iron store ferritin. 

Treatment of DNA with iron chelators such as EDTA, DETAP AC or desferrioxamine protects it from degradation. Thus, the destruction of DNA by bleomycin is iron-dependent. The breakdown of bleomycin may be initiated by a ferryl radical species or a hydroxyl... 

H6H, which has been cloned and expressed heterologously (162), is a nonheme, iron-dependent, oxoglutarate-dependent protein. It seems that the epoxidation reaction occurs much more slowly than the hydroxylation reaction. The tropane alkaloids seem to be formed in the roots and then transported to the aerial parts of the plant (163). 

Another subgroup of the 2His-lcarboxylate family is dependent on a reduced pterin cofactor (5). They catalyze hydrox-ylations at the aromatic positions of amino acids in phenylalanine catabolism and hormone biosynthesis (Fig. 2). Unlike the a-KG-dependent enzymes, the pterin co-substrate does not ligate to the iron directly. In the reaction cycle, the pterin cosubstrate supplies two electrons for the heteiolysis of O2 to give a yet to be characterized iron-oxygen hydroxylating species. 

NO was shown by Kanner et al. [120] to inhibit iron-catalysed oxidation reactions by binding to ferrous complexes. It was also shown that NO inhibited the superoxide driven Fenton reaction which, in the presence of iron, generates hydroxyl radical (OH) in vitro. By adding varying amounts of NO to a Fenton reaction process the hydroxylation of benzoic acid was reduced. This demonstrates that depending on the fluxes of the different reactive species, NO may have an antioxidant capability. 

In the next step of the reaction cycle, the enzyme-product compound (T) is formed by radical recombination of the iron-bound hydroxyl radical and the substrate radical. Finally, the product is released in the rate-limiting step (31) of the reaction. This step is also dependent on MMOB, and the cycle is complete. It is possible that compounds P, K, Q, and R all have an additional oxygen-derived bridge in addition to the hydroxo bridge. Thus, compounds P and K would have one /i-peroxo and one /a-hydroxo bridge, and compounds Q and R could have one fi-hydroxo and one ix-oxo bridge each [cf (122b, 10, 31)]. 

The other major class of nonheme iron-dependent dioxygenases are the a-ketoglutarate-dependent dioxygenases, which catalyze the oxidative decarboxylation of cosubstrate a-ketoglutarate to form succinate and an iron(IV)-oxo intermediate, which is then used to carry out a range of hydroxylation, desaturation, and other oxidative reactions. While the majority of reactions catalyzed by this family of enzymes are involved in biosynthetic pathways, enzymes such as HPPD (see Section 8.16.2.1) are involved in degradation pathways, therefore it is appropriate to discuss this family of enzymes, and contrast them with the nonheme iron-dependent dioxygenases described in Section 8.16.1. 

A large family of these enzymes is now known, and their enzymology and structures have been reviewed. A number of crystal structures have been obtained for enzymes in this family, and in each case the mononuclear iron(II) center is coordinated by a His,His,Glu motif, also observed in the extradiol catechol dioxygenases, and in other nonheme iron-dependent enzymes. Structural studies on clavaminic acid synthase have indicated the structural basis for the separate hydroxylation and oxidative cyclization/ desaturation reactions catalyzed by this enzyme. ... 

Microsomal cytochrome P450 is an iron-dependent oxygenase which reduces one atom of oxygen to water whilst the other is transferred to the substrate. The general outcome of this reaction is hydroxylation of the substrate which may be important for its consequent breakdown or solubilisation if it is toxic. 

Precedents for Selective C-H Hydroxylations at Non-Heme Iron-Dependent... 

Ascorbic acid is required for many hydroxylase enzymes in the human body. Ascorbic acid is needed for conversion of tyrosine to the neurotransmitter dopamine and further hydroxylation to adrenaline and noradrenaline, for synthesis of carnitine from lysine, and probably for hydroxylation of steroid hormones. Ascorbate is also known to participate in hydroxylation of aromatic drugs and carcinogens via microsomal mono-oxygenase systems of Uver endoplasmic reticulum (31,32). Its role in the formation of collagen is thought to be to maintain iron in its ferrous state for an iron-dependent proline hydroxylase, or to act as a direct source of electrons for reduction of O2 (31). 

The existence of mitochondrial cytochrome P450 in adrenal cortex was reported originally by Harding et al (1964) and confirmed by subsequent studies. Adrenal cortical mitochondria catalyze a number of NADPH- and molecular-oxygen-dependent hydroxylation reactions that contribute to the biosynthesis of corticosteroids. The enzyme system for 11 jS-hydroxyla-tion has been isolated, and a successful reconstitution of its activity has been achieved by the interaction of three proteins, namely an NADPH-dependent flavoprotein (adrenodoxin reductase), an iron-sulfur protein (adrenodoxin), and the heme protein (cytochrome P450) that serves as the terminal oxidase for the electron transport system from NADPH to oxygen (Wang and... 

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